Nanolipogel vehicles for controlled delivery of different pharmaceutical agents

ABSTRACT

A “nanolipogel” is a delivery vehicle including one or more lipid layer surrounding a hydrogel core, which may include an absorbent such as a cyclodextrin or ion-exchange resin. Nanolipogels can be constructed so as to incorporate a variety of different chemical entities that can subsequently be released in a controlled fashion. These different incorporated chemical entities can differ dramatically with respect to size and composition. Nanolipogels have been constructed to contain co-encapsulated proteins as well as small hydrophobic drugs within the interior of the lipid bilayer. Agents incorporated within nanolipogels can be released into the milieu in a controlled fashion, for example, nanolipogels provide a means of achieving simultaneous sustained release of agents that differ widely in chemical composition and molecular weight. Additionally, nanolipogels can favorably modulate biodistribution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending application U.S.application Ser. No. 15/923,139, filed Mar. 16, 2018, which is acontinuation of U.S. application Ser. No. 15/155,055, filed May 15,2016, which is a continuation-in-part of U.S. application Ser. No.14/394,161, filed Oct. 13, 2014, which is a U.S. national phaseapplication filed under 35 U.S.C. § 371 claiming benefit to PCTInternational Patent Application No PCT/US2013/036487, filed Apr. 12,2013, which claims the benefit of and priority to U.S. ProvisionalApplication No. 61/623,486, filed Apr. 12, 2012, U.S. ProvisionalApplication No. 61/747,624, filed Dec. 31, 2012, and U.S. ProvisionalApplication No. 61/747,614, filed Dec. 31, 2012, each of whichdisclosures is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under AgreementsR01-HL085416 and R10-EB008260 awarded to Tarek M. Fahmy by the NationalInstitutes of Health, Public Health Grant Number HL-55397 awarded toTarek M. Fahmy, and NIRT Grant Number CTS3090609326 awarded to Tarek M.Faluny by the National Science Foundation. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention is generally in the field of sustained delivery ofhigh and low molecular weight, or hydrophilic and hydrophobic molecules,especially antigen or tumor and immunomodulatory molecules, usingcore-shell nanoparticulates, which may be targeted by size andcomposition to a desired cell or tissue type to enhance efficacy.

BACKGROUND OF THE INVENTION

Although efficacy of therapeutic treatments is critically dependent uponmechanism of action of the agent(s) used, other factors are ofteninstrumental in eliciting an optimal response. Tolerable dose and timeof administration relative to onset of disease are other keyconsiderations.

Additionally, there are a number of complex issues involvingpharmacokinetic and pharmacodynamic characteristics that can also besignificant in therapeutic response. Over the years many studies havebeen carried out with a vast array of therapeutic agents in an effort toestablish optimal strategies for drug delivery. Over time, more and moredrug regimens for virtually all types of diseases have been designed toinvolve combination therapies; in some instances, combinations are usedto improve efficacy by combining drugs that have the same or differentdisease targets; in others, drugs with different mechanisms of actionmay act synergistically; and in still others, combination therapiesmight involve one or more drugs that act directly on the disease statetogether with one or more agents that have a beneficial effect, such asreduction of pain and/or protection from side effects of organs notdirectly involved in the disease and/or promotion of desirableactivities by natural defensive mechanisms, notably the immune system.Such disparate drugs with disparate roles in disease treatment oftendiffer dramatically with respect to chemical nature and thus drugdelivery issues in combination therapy can be very challenging.

Strategies involving the use of miniaturized vehicles that canencapsulate drugs in such a way as to allow for controlled release haveshown promise as a way to optimize drug delivery characteristics. Suchsystems offer the possibility of successful treatment and control ofmany diseases with drugs whose systemic half-lives and biodistributionare critical. Because of the diverse chemical nature of different drugs,there is a distinct advantage in the design and availability of aminiaturized vehicle that can usefully control drug release in a mannerthat is agnostic to the chemical nature of the drug.

Particulate vaccines are promising technologies for creation of tunableprophylactics and therapeutics against a wide variety of conditions.Vesicular and solid biodegradable polymer platforms, exemplified byliposomes and polyesters, respectively, are two of the most ubiquitousplatforms in vaccine delivery studies. Immunization withpoly(lactide-co-glycolide) (PLGA) nanoparticles elicits prolongedantibody titers compared to liposomes and alum. The magnitude of thecellular immune response is highest in animals vaccinated with PLGA,which also shows a higher frequency of effector-like memory T-cellphenotype, leading to an effective clearance of intracellular bacteria.The difference in performance of these two common particulate platformsis shown not to be due to material differences but appears to beconnected to the kinetics of antigen delivery. Liposomes are easilymodified for encapsulation of small hydrophilic molecules, and evenproteins. However, the stability of these formulations and the releaseprofiles of encapsulated agents are not easily controlled. Biodegradablesolid particles, on the other hand, such as those fabricated frompoly(lactic-co-glycolic acid) (PLGA), are highly stable and havecontrollable release characteristics, but pose complications for facileencapsulation and controlled release of therapeutic cytokines or forcombinatorial delivery. To overcome these limitations, hybrid platformsthat integrate features of different materials can offer advantages incombinatorial encapsulation and delivery. Such systems have beendemonstrated based on a core-shell methodology in which an organic orinorganic mesoporous or nanoporous core entrapping molecules of interestis coated with lipids or polymers. These hybrid systems can enhanceencapsulation and release of a wide variety of agents, such as smallmolecule drugs, proteins and nucleic acids, while promoting favorablepharmacokinetics and biodistribution of the encapsulant.

Hybridized systems, as such, are clearly attractive drug deliveryalternatives and have been explored in different studies. Such systemscan be engineered with a fluid biological bilayer that enhances theircirculation or potential for targeting while enabling the delivery ofagents of different physical properties. Several core-shell hybridsystems have been demonstrated for this purpose and indeed offerexciting new possibilities for combinatorial delivery that can work incancer therapy.

It is clear that the rate of release of bioactives, especially in thevaccine field, is critically important to the function, not just whichbioactives are incorporated. The complexity associated with delivery oftwo different agents, such as an antigen and an immunomodulator, makesit more difficult to find a delivery vehicle that allows for controlledrelease of the agents at different rates. This is particularly the casewhere the properties of the two agents are different, such as when oneis hydrophobic and one is hydrophilic, or one is high molecular weightand the other is low molecular weight. Even though it is possible toprovide particles that differ in chemical properties, it is difficult toensure that the agents are released at the correct time, for example,without having to diffuse from the core through the shell, where thecore is hydrophobic and the shell is hydrophilic (or vice versa) and theproperties of the agents lead them to migrate into another area of thedelivery device rather than out of the device, or, for example, whereone agent is very low molecular weight and tends to diffuse out rapidlyand the other agent is very high molecular weight and tends to diffuseout extremely slowly.

It is therefore an object of the present invention to provide means fordelivery of two or more pharmaceutical agents at different rates,especially agents with different chemical properties and/or molecularweights.

SUMMARY OF THE INVENTION

Nanolipogels, methods of incorporation of agents into these deliveryvehicles and making and using these compositions for the treatment ofdisease have been developed. These are designed to be loaded with agentseither prior to, during or after formation and subsequently function ascontrolled-release vehicles for the agents. “Nanolipogel”, is ananoparticle that combines the advantages of both liposomes andpolymer-based particles for sustained delivery of both proteins andsmall molecules. In a preferred embodiment, the nanolipogel is aLipid-Enveloped Dendrimer (LED).

The nanolipogel is typically loaded with more than one agent such thatcontrolled release of the multiplicity of agents is subsequentlyachieved.

The nanolipogel is loaded with one or more first agents during formationand one or more second agents following formation by the process ofrehydration of the nanolipogel in the presence of the second agents. Forexample, the nanolipogel is loaded with a molecule that serves as anadjuvant and the nanolipogel thereafter incorporates one or more targetantigens after formation, for the controlled release of adjuvanttogether with the antigens. Alternatively, the nanolipogel loaded withadjuvant is inserted into the site of a tumor in a patient, the tumor isablated, the nanolipogel is loaded with released tumor antigens and thenanolipogel releases the tumor antigens together with adjuvant into thebody of the patient in a controlled manner.

A nanolipogel is constructed of any of several suitable hydrogelmaterials, or combinations thereof, or other materials that are capableof in situ loading and release of agent such as cyclodextrin or ionexchange resins. The nanolipogel can be in the form of spheres, discs,rods or other geometries with different aspect ratios. The nanolipogelis typically formed of synthetic or natural polymers capable ofencapsulating agents by remote loading and tunable in properties so asto facilitate different rates of release.

A nanolipogel is a delivery vehicle including a lipid bilayersurrounding a hydrogel core, which may include an absorbent such as acyclodextrin or ion-exchange resin. Nanolipogels can be constructed soas to incorporate a variety of different chemical entities that cansubsequently be released in a controlled fashion. These differentincorporated chemical entities can differ dramatically with respect tosize and composition. Nanolipogels have been constructed to containco-encapsulated proteins as well as small hydrophobic drugs within theinterior of the lipid bilayer. Agents incorporated within nanolipogelscan be released into the milieu in a controlled fashion, for example,nanolipogels provide a means of achieving simultaneous sustained releaseof agents that differ widely in chemical composition and molecularweight. Additionally, nanolipogels can favorably modulatebiodistribution.

In a non-limiting example, one of the agents is an antigen and a secondagent is an immunoadjuvant, resulting in sustained release of theantigen together with the adjuvant to optimize an immune response. Inone example, simultaneous sustained delivery by nanolipogels of animmunostimulatory protein, Interleukin-2 (IL-2), as well as a lowmolecular weight organic molecule,2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride, an inhibitor of transforming growth factor-β (TGF-β), isachieved. This construct leads to an anti-tumor response in a murinesystem that is far superior to that achievable with the administrationin solution of either agent alone or a combination of the two.

The examples demonstrate the importance of antigen persistence mediatedby particulate platforms and its role in the long-term appearance ofeffector memory cellular response. Systemic administration ofCD4-targeted cytokine-loaded nanoparticles (“NPs”) was able to promotetolerance through expansion of host regulatory cells in murine allograftmodels. CD4-targeted TGF-β/IL-2 NPs alone induced a 3% increase in Tregfrequency in the spleen and mesenteric lymph nodes of healthy mice.Donor-specific transfusion of splenocytes pretreated with CD4-targetedLIE nanoparticles NPs resulted in a 4-fold increase in donor-specificTregs and significantly enhanced tolerance of fully mismatched heartallografts from 7 to 12 days.

In the B16 murine melanoma model, the proliferative and antitumoreffects of IL-2 cytokine therapy are hypothesized to be thwarted byTGF-β secretion and the activity of Tregs within the tumor. Therefore,nanolipogels were used to co-deliver the small molecule TGF-β inhibitorSB505124 and IL-2 to the tumor microenvironment. Tumor-localized drugdelivery was found to be critical. The most striking and significantsurvival benefits or reductions in tumor growth were observed in micereceiving simultaneous Nanolipogel (“nLG”)-mediated delivery of SB andIL-2. Monotherapies and soluble agent therapy failed to delaysubcutaneous tumor growth even when administered via intratumoralinjection, but nLG-SB+IL-2 induced significant delays in tumor growthrates and even resulted in complete regression in 40% of mice followedfor long-term survival. Encapsulation in nanolipogels improved drugcirculation following intravenous dosing and resulted in a nearly 4-folddose increase in the lungs up to 72 hours after administration. Afterone week of therapy, intravenous Nanolipogel (nLG-SB+IL-2) therapysignificantly reduced the number of metastatic lung growths compared tocontrols.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are schematics of the fabrication of the nanolipogelparticles (nLG). In FIG. 1A, methacrylate-functionalized cyclodextrin(CD) was used to solubilize a bioactive such as the TGF-β inhibitor(SB505124). In FIG. 1B, nanolipogels were formulated from lyophilizedliposomes loaded with biodegradable crosslinking polymer, acrylated-drug(CD-SB505) complex, and a second drug such as the peptide IL-2 cytokine.This core-shell structure facilitated entrapment of drug loaded CD andthe IL-2 in an interior biodegradable polymer matrix with a PEGylatedliposomal exterior. Succinylated β-cyclodextrin (CTD, Inc.) wasfunctionalized with 2-aminoethyl methacrylate (Sigma) by stirring a 1:3molar ratio of the compounds in 1×PBS for 1 hour at room temperature.The ¹H NMR spectra (500 MHz, D₂O) of SB505124, randomly succinylatedβ-CD, and the inclusion complex of SB505124 with randomly succinylatedβ-CD was determined. The differences observed in the aromatic protonregion of SB505124 demonstrate formation of the inclusion complex. The¹H NMR spectra (500 MHz, D₂O) of rhodamine B, randomly succinylatedβ-CD, and the inclusion complex of rhodamine B with randomlysuccinylated β-CD showed the differences observed in the aromatic protonregion of rhodamine B demonstrate formation of the inclusion complex.

FIGS. 1C-1G show nanolipogel characterization. Nanolipogel size wasdetermined by dynamic light scattering on a ZetaPALS instrument(Brookhaven Instruments) in PBS at room temperature. FIG. 1C shows thatencapsulation of SB or SB+IL-2 had no significant effect on particlemean diameter or polydispersity. Mean diameter and polydispersity indexare representative of 2 lots of each nanolipogel type (n=10 measurementsper sample). The zeta potential of PC/cholesterol liposomes,PC/cholesterol/PE-PEG-NH₂ liposomes, and nanolipogels were evaluated in0.1×PBS using a Malvern nanosizer. FIG. 1D shows that the zeta potentialof liposomes and nanolipogels incorporating amine-terminated PE-PEG wasfound to be close to neutral. FIG. 1E shows the composition andformulation properties of the nanolipogel formulation. FIG. 1F shows thepolymer structure verified by ¹H NMR. Cryo-TEM of nanolipogelsdemonstrate the formation of spherical liposomal structures. For TEManalysis, nanolipogel samples were stained with osmium tetroxide andthen imaged on an FEI Tenai Biotwin microscope. Lipid-specific osmiumtetroxide staining of cryosectioned samples had a localized stainingpattern confined to the exterior membrane of the particle. FIG. 1G showsthat the photopolymerized polymer/CD forms nanoparticulate hydrogelstructures that are detectable by light scattering even after disruptionof the liposomal exterior by detergent.

FIGS. 2A-2E are comparative release profiles from nLG, liposomes andsolid polymer nanoparticles (PLGA). Cumulative CD- or methacrylatefunctionalized-CD (f-CD)-solubilized SB released from nLGs normalized byinitial carrier mass demonstrated that polymerization of nanolipogelsimproved the sustained nature of SB release (FIG. 2A). Hydroxypropylβ-CD was used for SB complexation with the unfunctionalized CD.Cumulative IL-2 released determined by ELISA (immunoactive) and by abioactivity study (bioactive) from nLGs normalized by initialnanolipogel mass demonstrated that bioactivity of IL-2 was unaffected byencapsulation (FIG. 2B). Release of SB and IL-2 was not affected byincubation of 10 mg nLG in 1 ml full serum (FIG. 2C). Comparativecumulative release of SB from liposomes, nanolipogels, and degradablepolymeric (poly lactide-co-glycolide) nanoparticles (PLGA NPs)demonstrated that incorporation of photo-cured polymer in thenanolipogel vehicle enabled better sustained release and more completerelease of cyclodextrin-solubilized SB (FIG. 2D). PLGA NPs (meandiameter'² 150±50 nm) were prepared by using a modified water/oil/waterdouble emulsion technique. Liposomes were prepared in an identicalmanner as the nLG without the polymer core. Liposomes were loaded withIL-2 and SB similar to nanolipogels. The diminished percent ofencapsulated SB released from PLGA NPs is attributed to the interactionof the relatively hydrophobic polymer with SB. All particulateformulations were dissolved in 0.1N NaOH+1% SDS to determine 100%release at 7 days (arrow) (FIG. 2D). Comparative cumulative release ofIL-2 from liposomes, nanolipogels, and PLGA NPs demonstrated thatencapsulation of IL-2 in nanolipogels enabled better sustained releaseof cytokine. Cumulative release is presented as % of total IL-2 releasedthrough 7 days. (FIG. 2E) Data in all graphs represent mean oftriplicate samples ±1 standard deviation. FIG. 2F compares the sizes andloading of IL-2 and SB in PLGA, nanolipogels and liposomes.

FIGS. 3A-3G are graphs showing controlled release, clearance, andbiodistribution. The distribution of both nanolipogel carrier andencapsulated drug payload was investigated using dual-labeled NLG;fluorescein-labeled phosphoethanolamine was incorporated into the lipidcomponent of rhodamine-loaded nanolipogels. Spectrofluorimetric analysisat 540/625 nm and 490/517 nm show dose-dependent fluorescence with nospectral overlap.

FIG. 3A is a graph of cumulative IL-2 (ng/mg nLG) and drug (μg SB/mgnLG) released from co-loaded nLGs normalized by carrier mass. Error barsin all plots represent ±1 standard deviation. All experiments wererepeated at least twice with similar results. FIG. 3B is a graph showingclearance (percent of initial dose) of drug dose over time in days:Encapsulation in nanolipogels significantly increased the remainingpercentage of initial dose in the blood at 1 and 24 hours post-injection(two population t test, p<0.01 ###). FIG. 3C is a graph of whole bodydistribution. Mice received a single dose of rhodamine-loadednanolipogel or soluble rhodamine (in saline) via intravenous tail veininjection. Animals were sacrificed at 1, 24, 48, and 72 hourspost-injection for extraction and quantification of fluorescence; wholebody biodistribution was determined with rhodamine labeling.Significantly higher (two population t test, p<0.01) amounts ofrhodamine were detected in the major organs of nanolipogel-treatedanimals compared to animals injected with free dye. FIG. 3D is a graphof time dependent accumulation n in subcutaneous tumor: Cumulativerhodamine tumor penetration (circles) after B16 peritumoral injection inB6 mice. Peritumoral tissue was collected to quantify the remaining doseof nLG surrounding the tumor (squares). Controlled release demonstratesrelease of rhodamine, but not lipid (FIG. 3E). Mice bearing subcutaneousB16 tumors received a single IV (tail vein) injection of dual-labeledNLG. Animals were sacrificed at 1, 2, 3, and 7 days post injection andtissues collected for homogenization, extraction, and quantification ofrhodamine and fluorescein-PE. Analysis of serum shows prolongedcirculation of both encapsulant and delivery vehicle. Similar patternsof biodistribution were observed between lipid (FIG. 3F) and drugpayload (FIG. 3G), with highest accumulations of drug occurring in thelungs and liver.

FIGS. 4A-4C are graphs showing the clinical effects of intratumoralnanolipogel therapy on subcutaneous melanoma. FIG. 4A is a graph oftumor area versus time (day 0 was the day of tumor cell injection). Redarrows indicate treatments (via intratumoral injection). Mice bearingsubcutaneous tumors were euthanized when either greatest tumor dimensionwas larger than 15 mm or when exhibiting signs of illness. Tumor areasof deceased mice were not included after the day of death. Each groupinitially contained five mice except for the nLG-SB+IL-2 group, whichcontained four. Error bars represent ±1 standard deviation. Tumors inthe nLG-SB and nLG-SB+IL-2 groups are significantly smaller than allother groups (two population T test, P<0.001) beginning day 12. Tumorsin the nLG-SB+IL-2 group are significantly smaller than in the nLG-SBgroup starting day 17 (P<0.01, ##) and all other days afterwards(P<0.001). FIG. 4B is a graph of tumor masses of nLG-treated groupsseven days after treatment. Mice were euthanized directly prior to tumormass determination. Error bars represent ±1 standard deviation averagedacross six (nLG-Empty), ten (nLG-IL-2), nine (nLG-SB), and ten(nLG-SB+IL-2) mice. Each group initially contained 10 mice. Twopopulation T tests showed tumors in the nLG-SB+IL-2 group weresignificantly lower than the nLG-Empty (P<0.001, ***), nLG-IL-2(P<0.001, ***), and nLG-SB (P<0.05, *) groups. A two population T testshowed tumor masses in the nLG-IL-2 group were significantly lower thanthe nLG-Empty (P<0.05, #). FIG. 4C is a survival plot of mice from thesame study given in FIG. 4A. Arrows denote treatment days. The survivalof mice treated with nLG-SB was significantly longer by Mantel-Cox andGehan-Breslow-Wilcoxon analyses (P<0.01) and nLG-SB+IL-2 significantlyextended survival by both analyses (P<0.001). Studies were repeated 2-3times with similar results.

FIGS. 5A-5C shows the adaptive immune response and mechanism ofnLG-SB+IL-2 action. Each group contained six mice and studies wererepeated 2-3 times with similar results. FIG. 5A is a graph of theabsolute number of activated CD8⁺ cells present in lung tumors(normalized per number of tumors). All groups have significantly greaternumbers (P<0.01) compared with empty nLGs. FIG. 5B is a graph of theabsolute number of activated CD8⁺ cells present in tumors (normalizedper tumor mass) removed from mice seven days after treatment. Treatmentwith nLG-SB significantly increased activated CD8⁺ populations (P<0.05),as did treatment with nLG-IL-2 or nLG-SB+IL-2 (P<0.001), over unloadedparticles. Error bars represent ±1 standard deviation averaged acrosssix (nLG-Empty), ten (nLG-IL-2), nine (nLG-SB), and ten (nLG-SB+IL-2)mice. Each group initially contained 10 mice. FIG. 5C is a graph of theactivated CD8⁺:Treg ratio in TILs. All groups have significantly greaterratios (P<0.05) compared with empty nLGs.

FIGS. 6A-6C are graphs showing the role of NK cells in tumorimmunotherapy after combination delivery. Each group contained six miceand studies were repeated 2-3 times with similar results. FIG. 6A is agraph of the absolute number of NK cells present in tumors (normalizedper number of tumors). Compared to the empty particle group,significantly more NKs were present in the lungs following treatment bynLG-SB+IL-2 (P<0.05), nLG-SB (P<0.05), and nLG-IL-2 (P<0.01). FIG. 6B isa graph comparing tumor masses from wild type (WT) or NK-depleted (NKD)mice euthanized seven days after initial treatment. Each group initiallycontained 10 mice. The nLG-SB+IL-2-treated WT group has significantlysmaller tumors than all other treatment groups (P<0.001). The NKD nLG-SBand nLG-SB+IL-2 groups have significantly larger tumors than their WTcounterparts (both P<0.001). Studies were repeated 2-3 times withsimilar results. FIG. 6C is a graph of the absolute number of NK cellspresent in tumors (normalized per tumor mass) for the same study. ThenLG-SB+IL-2-treated group has significantly more NKs than the controlgroup (P<0.01), the SB-treated group (P<0.05), and the IL-2-treatedgroup (P<0.01). Error bars represent d 1 standard deviation averagedacross six (nLG-Empty), ten (nLG-IL-2), nine (nLG-SB), and ten(nLG-SB+IL-2) mice.

FIG. 7A is a schematic of LED preparation encapsulating siRNA/Dendrimerpolyplex and drug combinations, with covalent modification of the OuterShell with targeting antibodies or single chain variable fragments(scFv). FIG. 7B is a graph of the cytotoxicity of LED and LEDencapsulating the model drug methotrexate (MTX). Bars indicatesuccessive dilutions of LED or drug or combinations from 1 mg/ml to 10μg/ml. Azide is used as a positive control for cell killing. FIG. 7C isa bar graph showing the % cells exhibiting endosomal disruptionfollowing treatment with unmodified generation 4 PAMAM dendrimers (G4),or dendrimers conjugated to cyclodextrin molecules (CD) that substitutedand shielded primary amines with or without FCCP, a small moleculeionophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone. FIG. 7D isa bar graph showing the number of GFP positive cells as a percent oftotal cells transfected with pGFP using various LEDs (G4, G4-3CD,G4-6CD) at various N/P ratios. FIG. 7E is a bar graph showing relativenumber of MFICD3+, CD4+ cells control and various LEDs encapsulatingdifferent dosages of CD4 or Luciferase siRNA constructs. FIG. 7F is abar graph showing the level of GFP expression in 293T cells stablytransfected with eGFP following transfection of an siGFP construct usingLIPOFECTAMINE® or various LEDs containing combinations of differentdendrimer (G)-cyclodextrin conjugates (CDs). This graph measures themean fluorescence intensity (MFI) of GFP to assess silencing ability ofmodified dendrimers complexed with siGFP. The x-axis should read asfollows:

mock: nonsense siRNA

LFA: control siRNA against LFA

G3: unmodified generation 3 PAMAM dendrimer

G3 5×: G3 dendrimer with 1 cyclodextrin conjugated (G3-1CD)

G3 5×d: G3 with 2 CD conjugated (G3-2CD)

G3 10×: G3 with 3 CD conjugated (G3-3CD)

G3 20×: G3 with 3.4 CD conjugated (G3-3.4CD)

G4: G4 dendrimer with no modifications (G4)

G4 5×: G4 dendrimer with 1 CD conjugated (G4-1CD)

G4 5×d: G4 dendrimer with 1.3 CD conjugated (G4-1.3CD)

G4 10×: G4-3CD

G5: generation 5 (G5) dendrimer with no modifications

G5 5×: G5-1CD

G5 10×: G5-3CD

G5 10× 0.5 mg: G5-3CD, 500 ug used instead of 200 ug in other treatments

G5 10× D: G5-2.5CD

G5 20×: G5-4CD

FIG. 8A is a bar graph showing the % MHC-SIINFEKL, murinebone-marrow-derived dendritic cells (BMDCs). MHC-SINFEKL positive cellsfollowing treatment with liposomes containing ovalbumin alone (OVA),dendrimer alone, or a combination of OVA and dendrimer. * p<0.05 byone-way ANOVA Bonferroni post-test. FIG. 8B is a bar graph showing the %MHC-SINFEKL positive cells (by 25.D16-PE staining) with various controlsand liposomes including one or more of dendrimer (i.e., G5), antigen(i.e., ovalbumin (OVA)), and surface modifications (i.e., MPLA, and/orCpG) as labeled. The particle formulation containing MPLA, OVA, G5, andCpG was not shown since it encapsulated a prohibitively low amount ofOVA protein, and normalizing treatment groups by the amount of OVAresulted in cell toxicity because the particle concentration was higherthan other groups. FIG. 8C is a bar graph showing the IL-6 (pg/mL)expressed from bone marrow dendritic cells (BMDC) treated with LEDpresenting increasing amounts of CpG.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Nanolipogel,” as used herein, refers to a core-shell nanoparticlehaving a polymer matrix core, which can contain a host molecule, withina liposomal shell, which may be unilamellar or bilamellar, optionallycrosslinked.

“Host molecule,” as used herein, refers to a molecule or material whichreversibly associates with an active agent to form a complex. Inparticular embodiments, the host is a molecule that forms an inclusioncomplex with an active agent. Inclusion complexes are formed when anactive agent (i.e., the guest) or portion of an active agent insertsinto a cavity of another molecule, group of molecules, or material(i.e., the host). The host may be a small molecule, an oligomer, apolymer, or combinations thereof. Exemplary hosts includepolysaccharides such as amyloses, cyclodextrins, and other cyclic orhelical compounds containing a plurality of aldose rings, for example,compounds formed through 1,4 and 1,6 bonding of monosaccharides (such asglucose, fructose, and galactose) and disaccharides (such as sucrose,maltose, and lactose). Other exemplary host compounds include cryptands,cryptophanes, cavitands, crown ethers, dendrimers, ion-exchange resins,calixarenes, valinomycins, nigericins, catenanes, polycatenanes,carcerands, cucurbiturils, and spherands.

“Small molecule,” as used herein, refers to molecules with a molecularweight of less than about 2000 g/mol, more preferably less than about1500 g/mol, most preferably less than about 1200 g/mol.

“Hydrogel,” as used herein, refers to a water-swellable polymeric matrixformed from a three-dimensional network of macromolecules held togetherby covalent or non-covalent crosslinks, that can absorb a substantialamount of water (by weight) to form a gel.

“Nanoparticle”, as used herein, generally refers to a particle having adiameter from about 10 nm up to, but not including, about 1 micron,preferably from 100 nm to about 1 micron. The particles can have anyshape. Nanoparticles having a spherical shape are generally referred toas “nanospheres”.

“Molecular weight” as used herein, generally refers to the relativeaverage chain length of the bulk polymer, unless otherwise specified. Inpractice, molecular weight can be estimated or characterized usingvarious methods including gel permeation chromatography (GPC) orcapillary viscometry. GPC molecular weights are reported as theweight-average molecular weight (Mw) as opposed to the number-averagemolecular weight (Mn). Capillary viscometry provides estimates ofmolecular weight as the inherent viscosity determined from a dilutepolymer solution using a particular set of concentration, temperature,and solvent conditions.

“Mean particle size” as used herein, generally refers to the statisticalmean particle size (diameter) of the particles in a population ofparticles. The diameter of an essentially spherical particle may referto the physical or hydrodynamic diameter. The diameter of anon-spherical particle may refer preferentially to the hydrodynamicdiameter. As used herein, the diameter of a non-spherical particle mayrefer to the largest linear distance between two points on the surfaceof the particle. Mean particle size can be measured using methods knownin the art, such as dynamic light scattering.

“Monodisperse” and “homogeneous size distribution”, are usedinterchangeably herein and describe a population of nanoparticles ormicroparticles where all of the particles are the same or nearly thesame size. As used herein, a monodisperse distribution refers toparticle distributions in which 90% of the distribution lies within 15%of the median particle size, more preferably within 10% of the medianparticle size, most preferably within 5% of the median particle size.

“Active Agent”, as used herein, refers to a physiologically orpharmacologically active substance that acts locally and/or systemicallyin the body. An active agent is a substance that is administered to apatient for the treatment (e.g., therapeutic agent), prevention (e.g.,prophylactic agent), or diagnosis (e.g., diagnostic agent) of a diseaseor disorder.

II. Nanolipogels

Nanolipogels are core-shell nanoparticulates that combine the advantagesof both liposomes and polymer-based particles for sustained delivery ofactive agents. As discussed in more detail below, typically, the outershell protects cargo, provides biocompatibility and a surface forfunctionalization with targeting molecule(s). The outer shellencapsulates components so they are not exposed until desired, forexample, in response to environmental conditions or stimuli, creatingmonodisperse, reproducible particle populations, and mediatinginternalization into desired cell types. The inner core, which can be adendrimer or other polymer, has separate and additive functionalities toouter shell. For example, the inner shell allows for secondarydeposition of drug, vaccine, or imaging agent; increases loading ofcomponents with different physiochemical properties into the particle;allows for tunable release of contents from particles; increasescytosolic availability of DNA/RNA, drug, and/or protein by disruptingendosomes, all leading to enhanced drug effects, antigen presentation,and transfection/silencing.

Nanolipogels have a polymer matrix core containing one or more hostmolecules. The polymeric matrix is preferably a hydrogel, such as acrosslinked block copolymer containing one or more poly(alkylene oxide)segments, such as polyethylene glycol, and one or more aliphaticpolyester segments, such as polylactic acid. One or more host molecules,such as a cyclodextrin, dendrimer, or ion exchange resin, is dispersedwithin or covalently bound to the polymeric matrix. The hydrogel core issurrounded by a liposomal shell.

Nanolipogels can be constructed to incorporate a variety of activeagents that can subsequently be released in a controlled fashion. Activeagents can be dispersed within the hydrogel matrix, associated with oneor more host molecules, dispersed within the liposomal shell, covalentlyattached to the liposomal shell, and combinations thereof. Active agentscan be selectively incorporated at each of these locales within thenanolipogel. Furthermore, the release rate of active agents from each ofthese locales can be independently tuned. Because each of these localespossesses distinct properties, including size andhydrophobicity/hydrophilicity, the chemical entities independentlyincorporated at each of these locales can differ dramatically withrespect to size and composition. For example, nanolipogels can be loadedwith one or more proteins dispersed within the polymeric matrix as wellas small molecule hydrophobic drugs associated with host molecules.

In this way, nanolipogels can provide simultaneous sustained release ofagents that differ widely in chemical composition and molecular weight.In a non-limiting example, nanolipogels may be loaded with both ahydrophobic, small molecule antigen associated with a host molecule andan immunoadjuvant, such as an immunostimulatory protein, dispersedwithin the polymeric matrix. These nanolipogels can provide sustainedrelease of the antigen together with the adjuvant, so as to optimize animmune response. In a particular example, simultaneous sustaineddelivery by nanolipogels of an immunostimulatory protein, Interleukin-2(IL-2), as well as a low molecular weight organic molecule,2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride, an inhibitor of transforming growth factor-β (TGF-β), isachieved. This construct leads to an anti-tumor response in a murinesystem that is far superior to that achievable with the administrationin solution of either agent alone or a combination of the two.Additionally, nanolipogels can favorably modulate biodistribution of oneor more active agents encapsulated therein.

Nanolipogels are typically spherical in shape, with average particlesizes ranging between about 50 nm and about 1000 nm, more preferablybetween about 75 nm and about 300 nm, most preferably between about 90nm and about 200 nm. In certain embodiments, the nanolipogels possess anaverage particle size between about 100 nm and about 140 nm. Particlesmay be non-spherical.

Depending upon the nature of the lipids present in the liposomal shellof the nanolipogels, nanolipogels having a positive, negative, or nearneutral surface charge may be prepared. In certain embodiments, thenanolipogels possess a near neutral surface charge. In certainembodiments, the nanolipogels possess a ζ-potential of between about 10mV and about −10 mV, more preferably between about 5 mV and about −5 mV,more preferably between about 3 mV and about −3 mV, most preferablybetween about 2 mV and about −2 mV.

A. Core

The nanolipogel core is formed from a polymeric matrix and one or morehost molecules. The nanolipogel core may further include one or moreactive agents. The active agents may be complexed to the host molecules,dispersed with polymeric matrix, or combinations thereof.

1. Polymeric Matrix

The polymeric matrix of the nanolipogels may be formed from one or morepolymers or copolymers. By varying the composition and morphology of thepolymeric matrix, one can achieve a variety of controlled releasecharacteristics, permitting the delivery of moderate constant doses ofone or more active agents over prolonged periods of time.

The polymeric matrix may be formed from non-biodegradable orbiodegradable polymers; however, preferably, the polymeric matrix isbiodegradable. The polymeric matrix can be selected to degrade over atime period ranging from one day to one year, more preferably from sevendays to 26 weeks, more preferably from seven days to 20 weeks, mostpreferably from seven days to 16 weeks.

In general, synthetic polymers are preferred, although natural polymersmay be used. Representative polymers include poly(lactic acid),poly(glycolic acid), poly(lactic acid-co-glycolic acids),polyhydroxyalkanoates such as poly3-hydroxybutyrate orpoly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);poly(glycolide-co-caprolactones); polycarbonates such as tyrosinepolycarbonates; polyamides (including synthetic and natural polyamides),polypeptides, and poly(amino acids); polyesteramides; otherbiocompatible polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophilic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polysiloxanes; poly(oxyethylene)/poly(oxypropylene)copolymers; polyketals; polyphosphates; polyhydroxyvalerates;polyalkylene oxalates; polyalkylene succinates; poly(maleic acids),polyvinyl alcohols, polyvinylpyrrolidone; poly(alkylene oxides) such aspolyethylene glycol (PEG); derivativized celluloses such as alkylcelluloses (e.g., methyl cellulose), hydroxyalkyl celluloses (e.g.,hydroxypropyl cellulose), cellulose ethers, cellulose esters,nitrocelluloses, polymers of acrylic acid, methacrylic acid orcopolymers or derivatives thereof including esters, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate) (jointly referred to herein as“polyacrylic acids”), as well as derivatives, copolymers, and blendsthereof.

As used herein, “derivatives” include polymers having substitutions,additions of chemical groups and other modifications to the polymericbackbones described above routinely made by those skilled in the art.Natural polymers, including proteins such as albumin, collagen, gelatin,prolamines, such as zein, and polysaccharides such as alginate andpectin, may also be incorporated into the polymeric matrix. While avariety of polymers may be used to form the polymeric matrix, generally,the resulting polymeric matrix will be a hydrogel. In certain cases,when the polymeric matrix contains a natural polymer, the naturalpolymer is a biopolymer which degrades by hydrolysis, such as apolyhydroxyalkanoate.

In preferred embodiments, the polymeric matrix contains one or morecrosslinkable polymers. Preferably, the crosslinkable polymers containone or more photo-polymerizable groups, allowing for the crosslinking ofthe polymeric matrix following nanolipogel formation. Examples ofsuitable photo-polymerizable groups include vinyl groups, acrylategroups, methacrylate groups, and acrylamide groups. Photo-polymerizablegroups, when present, may be incorporated within the backbone of thecrosslinkable polymers, within one or more of the sidechains of thecrosslinkable polymers, at one or more of the ends of the crosslinkablepolymers, or combinations thereof.

The polymeric matrix may be formed from polymers having a variety ofmolecular weights, so as to form nanolipogels having properties,including drug release rates, optimal for specific applications.Generally, the polymers which make up the polymeric matrix possessaverage molecular weights of about 500 Da and 50 kDa. In cases where thepolymeric matrix is formed from non-crosslinkable polymers, the polymerstypically possess average molecular weights ranging between about 1 kDaand about 50 kDa, more preferably between about 1 kDa and about 70 kDa,most preferably between about 5 kDa and about 50 kDa. In cases where thepolymeric matrix is formed from crosslinkable polymers, the polymerstypically possess lower average molecular weights ranging between about500 Da and about 25 kDa, more preferably between about 1 kDa and about10 kDa, most preferably between about 3 kDa and about 6 kDa. Inparticular embodiments the polymeric matrix is formed from acrosslinkable polymer having an average molecular weight of about 5 kDa.

In some embodiments, the polymeric matrix is formed from a poly(alkyleneoxide) polymer or a block copolymer containing one or more poly(alkyleneoxide) segments. The poly(alkylene oxide) polymer or poly(alkyleneoxide) polymer segments may contain between 8 and 500 repeat units, morepreferably between 40 and 300 repeat units, most preferably between 50and 150 repeat units. Suitable poly(alkylene oxides) includepolyethylene glycol (also referred to as polyethylene oxide or PEG),polypropylene 1,2-glycol, poly(propylene oxide), polypropylene1,3-glycol, and copolymers thereof.

In some embodiments, the polymeric matrix is formed from an aliphaticpolyester or a block copolymer containing one or more aliphaticpolyester segments. Preferably the polyester or polyester segments arepoly(lactic acid) (PLA), poly(glycolic acid) PGA, orpoly(lactide-co-glycolide) (PLGA).

In preferred embodiments, the polymeric matrix is formed from a blockcopolymer containing one or more poly(alkylene oxide) segments, one ormore aliphatic polyester segments, and optionally one or morephoto-polymerizable groups. In these cases, the one or morepoly(alkylene oxide) segments imbue the polymer with the necessaryhydrophilicity, such that the resultant polymer matrix forms a suitablehydrogel, while the polyester segments provide a polymeric matrix withtunable hydrophobicity/hydrophilicity and/or the desired in vivodegradation characteristics.

The degradation rate of the polyester segments, and often thecorresponding drug release rate, can be varied from days (in the case ofpure PGA) to months (in the case of pure PLA), and may be readilymanipulated by varying the ratio of PLA to PGA in the polyestersegments. In addition, the poly(alkylene oxides), such as PEG, andaliphatic polyesters, such as PGA, PLA, and PLGA have been establishedas safe for use in humans; these materials have been used in humanclinical applications, including drug delivery system, for more than 30years.

In certain embodiments, the polymeric matrix is formed from a tri-blockcopolymer containing a central poly(alkylene oxide) segment, adjoiningaliphatic polyester segments attached to either end of the centralpoly(alkylene oxide) segment, and one or more photo-polymerizablegroups. Preferably, the central poly(alkylene oxide) segment is PEG, andaliphatic polyesters segments are PGA, PLA, or PLGA.

Generally, the average molecular weight of the central poly(alkyleneoxide) segment is greater than the average molecular weight of theadjoining polyester segments. In certain embodiments, the averagemolecular weight of the central poly(alkylene oxide) segment is at leastthree times greater than the average molecular weight of one of theadjoining polyester segments, more preferably at least five timesgreater than the average molecular weight of one of the adjoiningpolyester segments, most preferably at least ten times greater than theaverage molecular weight of one of the adjoining polyester segments.

In some cases, the central poly(alkylene oxide) segment possesses anaverage molecular weight ranging between about 500 Da and about 10,000Da, more preferably between about 1,000 Da and about 7,000 Da, mostpreferably between about 2,500 Da and about 5,000 Da. In particularembodiments, average molecular weight of the central poly(alkyleneoxide) segment is about 4,000 Da. Typically, each adjoining polyestersegment possesses an average molecular weight ranging between about 100Da and about 3,500 Da, more preferably between about 100 Da and about1,000 Da, most preferably between about 100 Da and about 500 Da.

In a preferred embodiment, the polymeric matrix is formed from thetri-block copolymer shown below

where m and n are, independently for each occurrence, integers between 1and 500, more preferably between 10 and 150.

Examples of preferred natural polymers include proteins such as albumin,collagen, gelatin and prolamines, for example, zein, and polysaccharidessuch as alginate, cellulose derivatives and polyhydroxyalkanoates, forexample, polyhydroxybutyrate. The in vivo stability of themicroparticles can be adjusted during the production by using polymerssuch as poly(lactide-co-glycolide) copolymerized with polyethyleneglycol (PEG). If PEG is exposed on the external surface, it may increasethe time these materials circulate due to the hydrophilicity of PEG.

Examples of preferred non-biodegradable polymers include ethylene vinylacetate, poly(meth)acrylic acid, polyamides, copolymers and mixturesthereof.

The matrix can also be made of gel-type polymers, such as alginate,produced through traditional ionic gelation techniques. The polymers arefirst dissolved in an aqueous solution, mixed with barium sulfate orsome bioactive agent, and then extruded through a microdroplet formingdevice, which in some instances employs a flow of nitrogen gas to breakoff the droplet. A slowly stirred (approximately 100-170 RPM) ionichardening bath is positioned below the extruding device to catch theforming microdroplets. The microparticles are left to incubate in thebath for twenty to thirty minutes in order to allow sufficient time forgelation to occur. Microparticle particle size is controlled by usingvarious size extruders or varying either the nitrogen gas or polymersolution flow rates. Chitosan microparticles can be prepared bydissolving the polymer in acidic solution and crosslinking it withtripolyphosphate. Carboxymethyl cellulose (CMC) microparticles can beprepared by dissolving the polymer in acid solution and precipitatingthe microparticle with lead ions. In the case of negatively chargedpolymers (e.g., alginate, CMC), positively charged ligands (e.g.,polylysine, polyethyleneimine) of different molecular weights can beionically attached.

Perhaps the most widely used are the aliphatic polyesters, specificallythe hydrophobic poly(lactic acid) (PLA), more hydrophilic poly(glycolicacid) PGA and their copolymers, poly(lactide-co-glycolide) (PLGA). Thedegradation rate of these polymers, and often the corresponding drugrelease rate, can vary from days (PGA) to months (PLA) and is easilymanipulated by varying the ratio of PLA to PGA. Second, the physiologiccompatibility of PLGA and its hompolymers PGA and PLA have beenestablished for safe use in humans; these materials have a history ofover 30 years in various human clinical applications including drugdelivery systems. PLGA nanoparticles can be formulated in a variety ofways that improve drug pharmacokinetics and biodistribution to targettissue by either passive or active targeting. The microparticles aredesigned to release molecules to be encapsulated or attached over aperiod of days to weeks. Factors that affect the duration of releaseinclude pH of the surrounding medium (higher rate of release at pH 5 andbelow due to acid catalyzed hydrolysis of PLGA) and polymer composition.Aliphatic polyesters differ in hydrophobicity and that in turn affectsthe degradation rate. Specifically the hydrophobic poly(lactic acid)(PLA), more hydrophilic poly (glycolic acid) PGA and their copolymers,poly(lactide-co-glycolide) (PLGA) have various release rates. Thedegradation rate of these polymers, and often the corresponding drugrelease rate, can vary from days (PGA) to months (PLA) and is easilymanipulated by varying the ratio of PLA to PGA.

2. Host Molecules

Host molecules are molecules or materials which reversibly associatewith an active agent to form a complex. By virtue of their ability toreversibly form complexes with active agents, host molecules canfunction to control the release of a complexed active agent in vivo.

In some cases, the host molecule is a molecule that forms an inclusioncomplex with an active agent. Inclusion complexes are formed when anactive agent (i.e., the guest) or portion of an active agent insertsinto a cavity of another molecule, group of molecules, or material(i.e., the host). Typically, the guest molecule associates with the hostmolecule without affecting the framework or structure of the host. Forexample, in the case of inclusion complexes, the size and shape of theavailable cavity in the host molecule remain substantially unaltered asa consequence of complex formation.

The host molecule may be a small molecule, an oligomer, a polymer, orcombinations thereof. Exemplary hosts include polysaccharides such asamyloses, cyclodextrins, and other cyclic or helical compoundscontaining a plurality of aldose rings, for example, compounds formedthrough 1,4 and 1,6 bonding of monosaccharides (such as glucose,fructose, and galactose) and disaccharides (such as sucrose, maltose,and lactose). Other exemplary host compounds include cryptands,cryptophanes, cavitands, crown ethers, dendrimers, ion-exchange resins,calixarenes, valinomycins, nigericins, catenanes, polycatenanes,carcerands, cucurbiturils, and spherands.

In still other embodiments, organic host compounds or materials includecarbon nanotubes, fullerenes, and/or grapheme-based host materials.Carbon nanotubes (CNTs) are allotropes of carbon with a cylindricalnanostructure. Nanotubes are members of the fullerene structural family,which also includes the spherical buckyballs, and the ends of a nanotubemay be capped with a hemisphere of the buckyball structure. Their nameis derived from their long, hollow structure with the walls formed byone-atom-thick sheets of carbon, called graphene. These sheets arerolled at specific and discrete (“chiral”) angles, and the combinationof the rolling angle and radius decides the nanotube properties.Nanotubes can be categorized as single-walled nanotubes (SWNTs) andmulti-walled nanotubes (MWNTs). Nanotubes and/or fullerenes can serve ashosts, for example, by encapsulating or entrapping the material to bedelivered (i.e., the guest) within the tubes or fullerenes.Alternatively, the exterior and/or interior of the tubes and/orfullerenes can be functionalized with functional groups which cancomplex to the guest to be delivered. Complexations include, but are notlimited to, ionic interactions, hydrogen bonding, Van der Waalsinteractions, and pi-pi interactions, such as pi-stacking.

Graphenes are also an allotrope of carbon. The structure of graphene isa one-atom-thick planar sheet of sp²-bonded carbon atoms that aredensely packed in a honeycomb crystal lattice. Graphene is the basicstructural element of some carbon allotropes including graphite,charcoal, carbon nanotubes and fullerenes. The guest to be delivered canassociate with and/or complex to graphene or functionalized graphene asdescribed above for nanotubes and fullerenes.

The host material can also be an inorganic material, including but notlimited to, inorganic phosphates and silica.

Suitable host molecules are generally selected for incorporation intonanolipogels in view of the identity of the active agent(s) to bedelivered and the desired drug release profile. In order to form acomplex with the active agent being delivered, the host molecule isgenerally selected to be complimentary to the active agent both in termsof sterics (size) and electronics (charge and polarity). For example, inthe case of host molecules that form inclusion complexes with the activeagent to be delivered, the host molecule will typically possess anappropriately-sized cavity to incorporate the active agent. In addition,the host molecule typically possesses a cavity of appropriatehydrophobicity/hydrophilicity to promote complex formation with theactive agent. The strength of the guest-host interaction will influencethe drug release profile of the active agent from the nanolipogel, withstronger guest-host interactions generally producing more prolonged drugrelease.

Generally, the host molecules are dispersed within the polymeric matrixthat forms the nanolipogel core. In some cases, one or more hostmolecules are covalently coupled to the polymeric matrix. For example,the host molecules may be functionalized with one or more pendantreactive functional groups that react with the polymer matrix. Inparticular embodiments, the host molecules contain one or more pendantreactive functional groups that react with the polymer matrix tocrosslink the polymer matrix. Examples of suitable reactive functionalgroups include methacrylates, acrylates, vinyl groups, epoxides,thiiranes, azides, and alkynes.

In certain embodiments, the host molecule is a cyclodextrin.Cyclodextrins are cyclic oligosaccharides containing six(α-cyclodextrin), seven (β-cyclodextrin), eight (γ-cyclodextrin), ormore α-(1,4)-linked glucose residues. The hydroxyl groups of thecyclodextrins are oriented to the outside of the ring while theglucosidic oxygen and two rings of the non-exchangeable hydrogen atomsare directed towards the interior of the cavity. As a result,cyclodextrins possess a hydrophobic inner cavity combined with ahydrophilic exterior. Upon combination with a hydrophobic active agent,the active agent (i.e., the guest) inserts into the hydrophobic interiorof the cyclodextrin (i.e., the host).

The cyclodextrin may be chemically modified such that some or all of theprimary or secondary hydroxyl groups of the macrocycle, or both, arefunctionalized with one or more pendant groups. The pendant groups maybe reactive functional groups that can react with the polymeric matrix,such as methacrylates, acrylates, vinyl groups, epoxides, thiiranes,azides, alkynes, and combinations thereof. The pendant groups may alsoserve to modify the solubility of the cyclodextrin. Exemplary groups ofthis type include sulfinyl, sulfonyl, phosphate, acyl, and C₁-C₁₂ alkylgroups optionally substituted with one or more (e.g., 1, 2, 3, or 4)hydroxy, carboxy, carbonyl, acyl, oxy, and oxo groups. Methods ofmodifying these alcohol residues are known in the art, and manycyclodextrin derivatives are commercially available.

Examples of suitable cyclodextrins include α-cyclodextrin;β-cyclodextrin; γ-cyclodextrin; methyl α-cyclodextrin; methylβ-cyclodextrin; methyl γ-cyclodextrin; ethyl β-cyclodextrin; butylα-cyclodextrin; butyl β-cyclodextrin; butyl γ-cyclodextrin; pentylγ-cyclodextrin; hydroxyethyl β-cyclodextrin; hydroxyethylγ-cyclodextrin; 2-hydroxypropyl α-cyclodextrin; 2-hydroxypropylβ-cyclodextrin; 2-hydroxypropyl γ-cyclodextrin; 2-hydroxybutylβ-cyclodextrin; acetyl ca-cyclodextrin; acetyl β-cyclodextrin; acetylγ-cyclodextrin; propionyl β-cyclodextrin; butyryl β-cyclodextrin;succinyl α-cyclodextrin; succinyl β-cyclodextrin; succinylγ-cyclodextrin; benzoyl β-cyclodextrin; palmityl β-cyclodextrin;toluenesulfonyl β-cyclodextrin; acetyl methyl β-cyclodextrin; acetylbutyl β-cyclodextrin; glucosyl α-cyclodextrin; glucosyl β-cyclodextrin;glucosyl γ-cyclodextrin; maltosyl α-cyclodextrin; maltosylβ-cyclodextrin; maltosyl γ-cyclodextrin; α-cyclodextrincarboxymethylether; β-cyclodextrin carboxymethylether; γ-cyclodextrincarboxymethylether; carboxymethylethyl β-cyclodextrin; phosphate esterα-cyclodextrin; phosphate ester β-cyclodextrin; phosphate esterγ-cyclodextrin; β-trimethylammonium-2-hydroxypropyl β-cyclodextrin;sulfobutyl ether β-cyclodextrin; carboxymethyl α-cyclodextrin;carboxymethyl β-cyclodextrin; carboxymethyl γ-cyclodextrin, andcombinations thereof.

Preferred cyclodextrins include α-cyclodextrins, β-cyclodextrins, andγ-cyclodextrins functionalized with one or more pendant acrylate ormethacrylate groups. In a particular embodiment, the host molecule is aβ-cyclodextrin functionalized with multiple methacrylate groups. Anexemplary host molecule of this type is illustrated below, wherein Rrepresents a C₁-C₆ alkyl group.

As a further example, the host molecule may also be a material thattemporarily associates with an active agent via ionic interactions. Forexample, conventional ion exchange resins known in the art for use incontrolled drug release may serve as host molecules. See, for example,Chen, et al. “Evaluation of ion-exchange microspheres as carriers forthe anticancer drug doxorubicin: in vitro studies.” J. Pharm. Pharmacol.44(3):211-215 (1992) and Farag, et al. “Rate of release of organiccarboxylic acids from ion exchange resins” J. Pharm. Sci.77(10):872-875(1988).

By way of exemplification, when the active agent being delivered is acationic species, suitable ion exchange resins may include a sulfonicacid group (or modified sulfonic acid group) or an optionally modifiedcarboxylic acid group on a physiologically acceptable scaffold.Similarly, where the active agent is an anionic species, suitable ionexchange resins may include amine-based groups (e.g., trimethylamine fora strong interaction, or dimethylethanolamine for a weaker interaction).Cationic polymers, such as polyethyleneimine (PEI), can function as hostmolecules for complex oligonucleotides such as siRNA.

In other cases, the host molecule is a dendrimer, such as apoly(amidoamine) (PAMAM) dendrimer. Cationic and anionic dendrimers canfunction as host materials by ionically associating with active agents,as described above. In addition, medium-sized dendrimers, such as three-and four-generation PAMAM dendrimers, may possess internal voids spaceswhich can accommodate active agents, for example, by complexation ofnucleic acids.

In some embodiments the host molecule is a dendrimer conjugated to acyclodextrin. In some embodiments, the cyclodextrin(s) shields primaryamines of dendrimer. Suitable dendrimers and cyclodextrins are discussedabove. Unmodified dendrimer (i.e., generation 4 PAMAM dendrimer (G4))was empirically better at endosomal disruption than dendrimer conjugatedwith cyclodexrin (CD) (See the Examples below). Without being bound bytheory, it is believed that terminal amine groups on PAMAM dendrimersprovide endosomal buffering and disrupt endosomes by the proton spongeeffect. Accordingly, increasing CD results in a decrease in endosomaldisruption. As discussed in the Examples below, different combinationsof dendrimers and cyclodextrins can be used to modulate the transfectionefficiency and level of endosomal disruption in the cell.

Preferably, the one or more host molecules are present in an amount offrom about 0.1% to about 40% w/w of the polymeric matrix, morepreferably from about 0.1% to about 25% w/w of the overall formulation.

3. Active Agents

Active agents to be delivered include therapeutic, nutritional,diagnostic, and prophylactic agents. The active agents can be smallmolecule active agents or biomacromolecules, such as proteins,polypeptides, or nucleic acids. Suitable small molecule active agentsinclude organic and organometallic compounds. The small molecule activeagents can be a hydrophilic, hydrophobic, or amphiphilic compound.

Exemplary therapeutic agents that can be incorporated into nanolipogelsinclude tumor antigens, CD4+ T-cell epitopes, cytokines,chemotherapeutic agents, radionuclides, small molecule signaltransduction inhibitors, photothermal antennas, monoclonal antibodies,immunologic danger signaling molecules, other immunotherapeutics,enzymes, antibiotics, antivirals (especially protease inhibitors aloneor in combination with nucleosides for treatment of HIV or Hepatitis Bor C), anti-parasites (helminths, protozoans), growth factors, growthinhibitors, hormones, hormone antagonists, antibodies and bioactivefragments thereof (including humanized, single chain, and chimericantibodies), antigen and vaccine formulations (including adjuvants),peptide drugs, anti-inflammatories, immunomodulators (including ligandsthat bind to Toll-Like Receptors (including but not limited to CpGoligonucleotides) to activate the innate immune system, molecules thatmobilize and optimize the adaptive immune system, molecules thatactivate or up-regulate the action of cytotoxic T lymphocytes, naturalkiller cells and helper T-cells, and molecules that deactivate ordown-regulate suppressor or regulatory T-cells), agents that promoteuptake of nanolipogels into cells (including dendritic cells and otherantigen-presenting cells), nutraceuticals such as vitamins, andoligonucleotide drugs (including DNA, RNAs, antisense, aptamers, smallinterfering RNAs, ribozymes, external guide sequences for ribonucleaseP, and triplex forming agents).

Exemplary diagnostic agents include paramagnetic molecules, fluorescentcompounds, magnetic molecules, and radionuclides, x-ray imaging agents,and contrast agents.

In certain embodiments, the nanolipogel includes one or more anti-canceragents. Representative anti-cancer agents include, but are not limitedto, alkylating agents (such as cisplatin, carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine,carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites(such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosinearabinoside, fludarabine, and floxuridine), antimitotics (includingtaxanes such as paclitaxel and decetaxel and vinca alkaloids such asvincristine, vinblastine, vinorelbine, and vindesine), anthracyclines(including doxorubicin, daunorubicin, valrubicin, idarubicin, andepirubicin, as well as actinomycins such as actinomycin D), cytotoxicantibiotics (including mitomycin, plicamycin, and bleomycin),topoisomerase inhibitors (including camptothecins such as camptothecin,irinotecan, and topotecan as well as derivatives of epipodophyllotoxinssuch as amsacrine, etoposide, etoposide phosphate, and teniposide),antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®), other anti-VEGF compounds; thalidomide(THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®);endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors suchas sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib(Nexavar@), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib;transforming growth factor-α or transforming growth factor-β inhibitors,and antibodies to the epidermal growth factor receptor such aspanitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

In certain embodiments, the nanolipogel includes one or moreimmunomodulatory agents. Exemplary immunomodulatory agents includecytokines, xanthines, interleukins, interferons, oligodeoxynucleotides,glucans, growth factors (e.g., TNF, CSF, GM-CSF and G-CSF), hormonessuch as estrogens (diethylstilbestrol, estradiol), androgens(testosterone, HALOTESTIN® (fluoxymesterone)), progestins (MEGACE®(megestrol acetate), PROVERA® (medroxyprogesterone acetate)), andcorticosteroids (prednisone, dexamethasone, hydrocortisone).

Examples of immunological adjuvants that can be associated with theparticles include, but are not limited to, TLR ligands, C-Type LectinReceptor ligands, NOD-Like Receptor ligands, RLR ligands, and RAGEligands. TLR ligands can include lipopolysaccharide (LPS) andderivatives thereof, as well as lipid A and derivatives there ofincluding, but not limited to, monophosphoryl lipid A (MPL),glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4′-monophosphoryllipid A. In a specific embodiment, the immunological adjuvant is MPL. Inanother embodiment, the immunological adjuvant is LPS. TLR ligands canalso include, but are not limited to, TLR3 ligands (e.g.,polyinosinic-polycytidylic acid (poly(I:C)), TLR7 ligands (e.g.,imiquimod and resiquimod), and TLR9 ligands.

The nanolipogel may also include antigens and/or adjuvants (i.e.,molecules enhancing an immune response). Peptide, protein, and DNA basedvaccines may be used to induce immunity to various diseases orconditions. Cell-mediated immunity is needed to detect and destroyvirus-infected cells. Most traditional vaccines (e.g. protein-basedvaccines) can only induce humoral immunity. DNA-based vaccine representsa unique means to vaccinate against a virus or parasite because a DNAbased vaccine can induce both humoral and cell-mediated immunity. Inaddition, DNA based vaccines are potentially safer than traditionalvaccines. DNA vaccines are relatively more stable and morecost-effective for manufacturing and storage. DNA vaccines consist oftwo major components, DNA carriers (or delivery vehicles) and DNAsencoding antigens. DNA carriers protect DNA from degradation, and canfacilitate DNA entry to specific tissues or cells and expression at anefficient level.

In certain embodiments, the nanolipogel core contains two or more activeagents. In preferred embodiments, the nanolipogel core contains both asmall molecule hydrophobic active agent, preferably associated with oneor more suitable host molecules, and a hydrophilic active agentdispersed within the polymeric matrix. In particular embodiments, thehydrophilic active agent is a protein, such as a therapeutic cytokine.By incorporating a hydrophobic active agent in association with a hostmolecule and a hydrophilic molecule dispersed within the polymericmatrix, controlled release of two or more active agents, including twoor more active agents with varied physiochemical characteristics (suchas solubility, hydrophobicity/hydrophilicity, molecular weight, andcombinations thereof) can be achieved.

In a preferred embodiment demonstrated by the examples, the hostmolecule is used to deliver a low molecular weight compounds such as achemotherapeutic, where the host molecule retards release of the lowmolecular weight compound, and a larger hydrophilic compound, such as acytokine, so that release of both molecules occurs over a similar timeperiod.

B. Shell Components

Nanolipogels include a liposomal shell composed of one or moreconcentric lipid monolayers or lipid bilayers. The shell can furtherinclude one or active agents, targeting molecules, or combinationsthereof.

1. Lipids

Nanolipogels include a liposomal shell composed of one or moreconcentric lipid monolayers or lipid bilayers. The composition of theliposomal shell may be varied to influence the release rate of one ormore active agents in vivo. The lipids may also be covalentlycrosslinked, if desired, to alter in vivo drug release.

The lipid shell can be formed from a single lipid bilayer (i.e., theshell may be unilamellar) or several concentric lipid bilayers (i.e.,the shell may be multilamellar). The lipid shell may be formed from asingle lipid; however, in preferred embodiments, the lipid shell isformed from a combination of more than one lipid. The lipids can beneutral, anionic or cationic lipids at physiologic pH.

Suitable neutral and anionic lipids include sterols and lipids such ascholesterol, phospholipids, lysolipids, lysophospholipids, andsphingolipids. Neutral and anionic lipids include, but are not limitedto, phosphatidylcholine (PC) (such as egg PC, soy PC), including1,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS),phosphatidylglycerol, phosphatidylinositol (PI); glycolipids;sphingophospholipids, such as sphingomyelin, sphingoglycolipids (alsoknown as 1-ceramidyl glucosides), such as ceramide galactopyranoside,gangliosides and cerebrosides; fatty acids, sterols containing acarboxylic acid group such as cholesterol or derivatives thereof; and1,2-diacyl-sn-glycero-3-phosphoethanolamines, including1,2-dioleoyl-sn-Glycero-3-phosphoethanolamine or 1,2-dioleolylglycerylphosphatidylethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine(DHPE), 1,2-distearoylphosphatidylcholine (DSPC),1,2-dipalmitoylphosphatidylcholine (DPPC), and1,2-dimyristoylphosphatidylcholine (DMPC). Also suitable are natural(e.g., tissue derived L-α-phosphatidyl: egg yolk, heart, brain, liver,soybean) and/or synthetic (e.g., saturated and unsaturated1,2-diacyl-sn-glycero-3-phosphocholines,1-acyl-2-acyl-sn-glycero-3-phosphocholines,1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of theselipids.

Suitable cationic lipids includeN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, alsoreferred to as TAP lipids, for example as a methylsulfate salt. SuitableTAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP(dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Othersuitable cationic lipids include dimethyldioctadecyl ammonium bromide(DDAB), 1,2-diacyloxy-3-trimethylammonium propanes,N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP),1,2-diacyloxy-3-dimethylammonium propanes,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1,2-dialkyloxy-3-dimethylammonium propanes,dioctadecylamidoglycylspermine (DOGS),3-[N—(N′,N′-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol);2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminiumtrifluoro-acetate (DOSPA), β-alanyl cholesterol, cetyltrimethylammoniumbromide (CTAB), diC₁₄-amidine,N-tert-butyl-N′-tetradecyl-3-tetradecylamino-propionamidine,N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG),ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride,1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), andN,N,N′,N′-tetramethyl-,N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide,1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives, such as1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazoliniumchloride (DOTIM) and1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM), and 2,3-dialkyloxypropyl quaternary ammoniumderivatives containing a hydroxyalkyl moiety on the quaternary amine,for example, 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide(DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DORIE), 1,2-dioleyloxypropyl-3-dimetyl-hydroxypropyl ammonium bromide(DORIE-HP), 1,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammoniumbromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe),1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).

Other suitable lipids include PEGylated derivatives of the neutral,anionic, and cationic lipids described above. Incorporation of one ormore PEGylated lipid derivatives into the lipid shell can result in ananolipogel which displays polyethylene glycol chains on its surface.The resulting nanolipogels may possess increased stability andcirculation time in vivo as compared to nanolipogels lacking PEG chainson their surfaces. Examples of suitable PEGylated lipids includedistearoylphosphatidylethanlamine-polyethylene glycol (DSPE-PEG),including DSPE PEG (2000 MW) and DSPE PEG (5000 MW),dipalmitoyl-glycero-succinate polyethylene glycol (DPGS-PEG),stearyl-polyethylene glycol and cholesteryl-polyethylene glycol.

In preferred embodiments, the lipid shell is formed from a combinationof more than one lipid. In certain embodiments the lipid shell is formedfrom a mixture of at least three lipids. In particular embodiments, thelipid shell is formed from a mixture of phosphatidyl choline (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol.

In some embodiments, the lipid shell is formed from a mixture of one ormore PEGylated phospholipids and one or more additional lipids orsterols. In certain instances, the molar ratio of the one or morePEGylated lipids to the one or more additional lipids or sterols rangesfrom about 1:1 to about 1:6, more preferably from about 1:2 to about1:6, most preferably from about 1:3 to about 1:5. In particularembodiments, the molar ratio of the one or more PEGylated lipids to theone or more additional lipids or sterols is about 1:4.

In some embodiments, the lipid shell is formed from a mixture of one ormore phospholipids and one or more additional lipids or sterols. Incertain instances, the molar ratio of the one or more phospholipids tothe one or more additional lipids or sterols ranges from about 1:1 toabout 6:1, more preferably from about 2:1 to about 6:1, most preferablyfrom about 3:1 to about 5:1. In particular embodiments, the molar ratioof the one or more phospho lipids to the one or more additional lipidsor sterols is about 4:1.

In a preferred embodiments, the lipid shell is formed from a mixture ofa phospholipid, such as phosphatidyl choline (PC), a PEGylatedphospholipid, such as1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol. In particular embodiments,the lipid shell is formed from a mixture of phosphatidyl choline,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol in a 3:1:1 molar ratio.

2. Targeting Molecules and Molecules Decreasing RES Uptake

The surface of the nanolipogels, or the core host, can be modified tofacilitate targeting through the attachment of targeting molecules.Exemplary target molecules include proteins, peptides, nucleic acids,lipids, saccharides, or polysaccharides that bind to one or more targetsassociated with an organ, tissue, cell, or extracellular matrix, orspecific type of tumor or infected cell. The degree of specificity withwhich the nanolipogels are targeted can be modulated through theselection of a targeting molecule with the appropriate affinity andspecificity. For example, a targeting moiety can be a polypeptide, suchas an antibody that specifically recognizes a tumor marker that ispresent exclusively or in higher amounts on a malignant cell (e.g., atumor antigen). Suitable targeting molecules that can be used to directnanoparticles to cells and tissues of interest, as well as methods ofconjugating target molecules to nanoparticles, are known in the art.See, for example, Ruoslahti, et al. Nat. Rev. Cancer, 2:83-90 (2002).Targeting molecules can also include neuropilins and endothelialtargeting molecules, integrins, selectins, and adhesion molecules.Targeting molecules can be covalently bound to nanolipogels using avariety of methods known in the art.

In certain embodiments, the liposomal shell includes one or morePEGylated lipids. The PEG, or other hydrophilic polyalkylene oxide,avoids uptake of the lipogels by the reticuloendothelial system (“RES”),thereby prolonging in vivo residence time.

The surface of the nanolipogels can be modified to facilitate targetingthrough the attachment of targeting molecules. These can be proteins,peptides, nucleic acid molecules, saccharides or polysaccharides thatbind to a receptor or other molecule on the surface of a targeted cell.The degree of specificity can be modulated through the selection of thetargeting molecule. For example, antibodies are very specific. These canbe polyclonal, monoclonal, fragments, recombinant, or single chain, manyof which are commercially available or readily obtained using standardtechniques. T-cell specific molecules and antigens which are bound byantigen presenting cells as well as tumor targeting molecules can bebound to the surface of the nanolipogel and/or to the host molecule. Thetargeting molecules may be conjugated to the terminus of one or more PEGchains present on the surface of the liposomal shell.

3. Active Agents

The shell of the nanolipogels may optionally contain one or more activeagents, including any of the active agents described above.

Hydrophobic active agents, such as proteins, may be covalently connectedto the surface of the nanolipogel, whereas hydrophilic active agents maybe covalently connected to the surface of the nanolipogel or dispersedwithin the liposomal shell. In certain embodiments, the liposomal shellincludes one or more PEGylated lipids. In these cases, one or moreactive agents may be conjugated to the terminus of one or more PEGchains present on the surface of the liposomal shell. In particularembodiments, one or more active agents are covalently connected to thesurface of the nanolipogel via a linking group that is cleaved inresponse to an external chemical or physical stimulus, such as a changein ambient pH, so as to trigger release of the active agent at a desiredphysiological locale.

III. Methods of Manufacture, Loading, and Pharmaceutical Compositions

A. Methods of Manufacture and Loading

A nanolipogel is a nanoparticle that combines the advantages of bothliposomes and polymer-based particles for sustained delivery of nucleicacids, proteins and/or small molecules. The nanolipogel can be in theform of spheres, discs, rods or other geometries with different aspectratios. The nanosphere can be larger, i.e., microparticles. Thenanolipogel is typically formed of synthetic or natural polymers capableof encapsulating agents by remote loading and tunable in properties soas to facilitate different rates of release. Release rates are modulatedby varying the polymer to lipid ratio from 0.05 to 5.0, more preferablyfrom 0.5 to 1.5.

Nanolipogels are designed to be loaded with agents either prior to,during or after formation and subsequently function ascontrolled-release vehicles for the agents. The nanolipogel can beloaded with more than one agent such that controlled release of themultiplicity of agents is subsequently achieved.

The nanolipogel is loaded with one or more first agents during formationand one or more second agents following formation by the process ofrehydration of the nanolipogel in the presence of the second agents. Forexample, the nanolipogel is loaded with a molecule that serves as anadjuvant and the nanolipogel thereafter incorporates one or more targetantigens after formation, for the controlled release of adjuvanttogether with the antigens. Alternatively, the nanolipogel loaded withadjuvant is inserted into the site of a tumor in a patient, the tumor isablated, the nanolipogel is loaded with released tumor antigens and thenanolipogel releases the tumor antigens together with adjuvant into thebody of the patient in a controlled manner.

In another embodiment, the nanolipogel is loaded with an antigen, amolecule that serves as an adjuvant and a targeting molecule for antigenpresenting cells, the nanolipogel is taken up by antigen presentingcells and the antigen is appropriately processed and presented toT-helper and cytotoxic T-cells to promote a cell-mediated immuneresponse.

In yet another embodiment, the nanolipogel loaded with a molecule thatserves as an adjuvant and a targeting molecule for antigen presentingcells is inserted into the site of a tumor in a patient, the tumor isablated and the nanolipogel is loaded with released tumor antigens, thenanolipogel is taken up by antigen presenting cells and the releasedtumor antigens are appropriately processed and presented to T-helper andcytotoxic T-cells to promote a cell-mediated immune response.

B. Pharmaceutical Compositions

Pharmaceutical compositions including nanolipogels are provided.Pharmaceutical compositions can be for administration by parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), transdermal (either passively or using iontophoresis orelectroporation), or transmucosal (nasal, vaginal, rectal, orsublingual) routes of administration or using bioerodible inserts andcan be formulated in dosage forms appropriate for each route ofadministration.

In some embodiments, the compositions are administered systemically, forexample, by intravenous or intraperitoneal administration, in an amounteffective for delivery of the compositions to targeted cells. Otherpossible routes include trans-dermal or oral.

In certain embodiments, the compositions are administered locally, forexample by injection directly into a site to be treated. In someembodiments, the compositions are injected or otherwise administereddirectly to one or more tumors. Typically, local injection causes anincreased localized concentration of the compositions which is greaterthan that which can be achieved by systemic administration. In someembodiments, the compositions are delivered locally to the appropriatecells by using a catheter or syringe. Other means of delivering suchcompositions locally to cells include using infusion pumps (for example,from Alza Corporation, Palo Alto, Calif.) or incorporating thecompositions into polymeric implants (see, for example, P. Johnson andJ. G. Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England:Ellis Horwood Ltd., 1987), which can effect a sustained release of thenanolipogels to the immediate area of the implant.

The nanolipogels can be provided to the cell either directly, such as bycontacting it with the cell, or indirectly, such as through the actionof any biological process. For example, the nanolipogels can beformulated in a physiologically acceptable carrier or vehicle, andinjected into a tissue or fluid surrounding the cell. The nanolipogelscan cross the cell membrane by simple diffusion, endocytosis, or by anyactive or passive transport mechanism.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, age, and general health of the recipient, will be able toascertain proper dosing. The selected dosage depends upon the desiredtherapeutic effect, on the route of administration, and on the durationof the treatment desired. Generally dosage levels of 0.001 to 10 mg/kgof body weight daily are administered to mammals. Generally, forintravenous injection or infusion, dosage may be lower. Generally, thetotal amount of the nanolipogel-associated active agent administered toan individual will be less than the amount of the unassociated activeagent that must be administered for the same desired or intended effect.

1. Formulations for Parenteral Administration

In a preferred embodiment the nanolipogels are administered in anaqueous solution, by parenteral injection. The formulation can be in theform of a suspension or emulsion. In general, pharmaceuticalcompositions are provided including effective amounts of one or moreactive agents optionally include pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.Such compositions can include diluents sterile water, buffered saline ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength; and optionally, additives such as detergents andsolubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to aspolysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations may be lyophilizedand redissolved/resuspended immediately before use. The formulation maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

2. Formulations for Topical and Mucosal Administration

The nanolipogels can be applied topically. Topical administration caninclude application to the lungs, nasal, oral (sublingual, buccal),vaginal, or rectal mucosa. These methods of administration can be madeeffective by formulating the shell with transdermal or mucosal transportelements. For transdermal delivery such elements may include chemicalenhancers or physical enhancers such as electroporation or microneedledelivery. For mucosal delivery PEGylation of the outer shell or additionof chitosan or other mucosal permeants or PH protective elements fororal delivery.

Compositions can be delivered to the lungs while inhaling and traverseacross the lung epithelial lining to the blood stream when deliveredeither as an aerosol or spray dried particles having an aerodynamicdiameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent® nebulizer(Mallinekrodt Inc., St. Louis, Mo.); the Acorn® II nebulizer (MarquestMedical Products, Englewood, Colo.); the Ventolin® metered dose inhaler(Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powderinhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkindall have inhalable insulin powder preparations approved or in clinicaltrials where the technology could be applied to the formulationsdescribed herein.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets, capsules, or lozenges. Oralformulations may include excipients or other modifications to theparticle which can confer enteric protection or enhanced deliverythrough the GI tract, including the intestinal epithelia and mucosa (seeSamstein, et al. Biomaterials. 29(6):703-8 (2008).

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations can includepenetration enhancers. Chemical enhancers and physical methods includingelectroporation and microneedles can work in conjunction with thismethod.

IV. Methods of Treatment

The methods of treatment typically include using nanolipogels loadedwith one or more active agents, to deliver the one or more active agentsinto cells, or to a cell's microenvironment. The methods typicallyinclude contacting the active agent-loaded nanolipogel with one morecells. The contacting can occur in vivo or in vitro.

Administration of a drug or other cargo to cells or a subject usingnanolipogels can be compared to a control, for example, delivery of thedrug or other cargo to cells or a subject using conventional deliverymethods such as free cargo/drug delivery, delivery using conventionalPLGA nanoparticles, or delivery using conventional liposomal methodssuch as LIPOFECTAMINE®. Nanolipogels can be used to deliver cargo totarget cells with increased efficacy compared to conventional deliverymethods. In some embodiments less cargo or drug is required whendelivered using nanolipogels compared to conventional delivery methodsto achieve the same or greater therapeutic benefit.

In some embodiments toxicity is reduced or absent compared toconventional delivery methods. For example, in some embodiments, whiteblood cell, platelet, hemoglobin, and hematocrit levels were withinnormal physiological ranges; no liver or renal toxicities are observed;body weight and serum concentrations for alkaline phosphatase, alaninetransferase, total bilirubin, and blood urea nitrogen are normal; orcombinations thereof following administration of loaded nanolipogels tothe subject.

A. In Vivo Methods

The disclosed compositions can be used in a method of delivering activeagents to cells in vivo. In some in vivo approaches, the compositionsare administered to a subject in a therapeutically effective amount. Asused herein, the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate one ormore symptoms of the disorder being treated or to otherwise provide adesired pharmacologic and/or physiologic effect. The precise dosage willvary according to a variety of factors such as subject-dependentvariables (e.g., age, immune system health, etc.), the disease, and thetreatment being effected.

1. Drug Delivery

The particles can be used to deliver an effective amount of one or moretherapeutic, diagnostic, and/or prophylactic agents to an individual inneed of such treatment. The amount of agent to be administered can bereadily determine by the prescribing physician and is dependent on theage and weight of the patient and the disease or disorder to be treated.

The particles are useful in drug delivery (as used herein “drug”includes therapeutic, nutritional, diagnostic and prophylactic agents),whether injected intravenously, subcutaneously, or intramuscularly,administered to the nasal or pulmonary system, injected into a tumormilieu, administered to a mucosal surface (vaginal, rectal, buccal,sublingual), or encapsulated for oral delivery. The particles may beadministered as a dry powder, as an aqueous suspension (in water,saline, buffered saline, etc), in a hydrogel, organogel, in capsules,tablets, troches, or other standard pharmaceutical excipient

The preferred embodiment is a dry powder rehydrated with the capsulantof interest in sterile saline or other pharmaceutically acceptableexcipient.

As discussed herein, compositions can be used to as delivery vehiclesfor a number of active agents including small molecules, nucleic acids,proteins, and other bioactive agents. The active agent or agents can beencapsulated within, dispersed within, and/or associated with thesurface of the nanolipogel particle. In some embodiments, thenanolipogel packages two, three, four, or more different active agentsfor simultaneous delivery to a cell.

2. Transfection

The disclosed compositions can be for cell transfection ofpolynucleotides. As discussed in more detail below, the transfection canoccur in vitro or in vivo, and can be applied in applications includinggene therapy and disease treatment. The compositions can be moreefficient, less toxic, or a combination thereof when compared to acontrol. In some embodiments, the control is cells treated with analternative transfection reagent such as LIPOFECTAMINE 2000.

The particular polynucleotide delivered by the nanolipogel can beselected by one of skill in the art depending on the condition ordisease to be treated. The polynucleotide can be, for example, a gene orcDNA of interest, a functional nucleic acid such as an inhibitory RNA, atRNA, an rRNA, or an expression vector encoding a gene or cDNA ofinterest, a functional nucleic acid a tRNA, or an rRNA. In someembodiments two or more polynucleotides are administered in combination.

In some embodiments, the polynucleotide encodes a protein. Exemplaryproteins include, for example, (a) angiogenic and other factorsincluding growth factors such as acidic and basic fibroblast growthfactors, vascular endothelial growth factor, endothelial mitogenicgrowth factors, epidermal growth factor, transforming growth factor αand β, platelet-derived endothelial growth factor, platelet-derivedgrowth factor, tumor necrosis factor-α, hepatocyte growth factor andinsulin-like growth factor; (b) cell cycle inhibitors such ascyclin-dependent kinases, thymidine kinase (“TK”), and other agentsuseful for interfering with cell proliferation; (c) bone morphogenicproteins (“BMP's”), including BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1),BMP-7 (OP-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14,BMP-15, and BMP-16. BMPs are typically dimeric proteins that can beprovided as homodimers, heterodimers, or combinations thereof, alone ortogether with other molecules. Alternatively, or in addition, moleculescapable of inducing an upstream or downstream effect of a BMP can beprovided. Such molecules include any of the “hedgehog” proteins, or theDNA's encoding them.

In some embodiments, the polynucleotide is not integrated into the hostcell's genome (i.e., remains extrachromosomal). Such embodiments can beuseful for transient or regulated expression of the polynucleotide, andreduce the risk of insertional mutagenesis. Therefore, in someembodiments, the nanolipogels are used to deliver mRNA ornon-integrating expression vectors that are expressed transiently in thehost cell.

In some embodiments, the polynucleotide is integrated into the hostcell's genome. For example, gene therapy is a technique for correctingdefective genes responsible for disease development. Researchers may useone of several approaches for correcting faulty genes: (a) a normal genecan be inserted into a nonspecific location within the genome to replacea nonfunctional gene. This approach is most common; (b) an abnormal genecan be swapped for a normal gene through homologous recombination; (c)an abnormal gene can be repaired through selective reverse mutation,which returns the gene to its normal function; (d) the regulation (thedegree to which a gene is turned on or off) of a particular gene can bealtered.

Gene therapy can include the use of viral vectors, for example,adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neuronal trophic virus, Sindbis and other RNAviruses, including these viruses with the HIV backbone. Also useful areany viral families which share the properties of these viruses whichmake them suitable for use as vectors. Typically, viral vectors contain,nonstructural early genes, structural late genes, an RNA polymerase IIItranscript, inverted terminal repeats necessary for replication andencapsidation, and promoters to control the transcription andreplication of the viral genome. When engineered as vectors, virusestypically have one or more of the early genes removed and a gene orgene/promoter cassette is inserted into the viral genome in place of theremoved viral DNA.

Gene targeting via target recombination, such as homologousrecombination (HR), is another strategy for gene correction. Genecorrection at a target locus can be mediated by donor DNA fragmentshomologous to the target gene (Hu, et al., Mol. Biotech., 29:197-210(2005); Olsen, et al., J. Gene Med., 7:1534-1544 (2005)). One method oftargeted recombination includes the use of triplex-formingoligonucleotides (TFOs) which bind as third strands tohomopurine/homopyrimidine sites in duplex DNA in a sequence-specificmanner. Triplex forming oligonucleotides can interact with eitherdouble-stranded or single-stranded nucleic acids.

Methods for targeted gene therapy using triplex-forming oligonucleotides(TFO's) and peptide nucleic acids (PNAs) are described in U.S. PublishedApplication No. 20070219122 and their use for treating infectiousdiseases such as HIV are described in U.S. Published Application No.2008050920. The triplex-forming molecules can also be tail clamp peptidenucleic acids (tcPNAs), such as those described in U.S. PublishedApplication No. 2011/0262406. Highly stable PNA:DNA:PNA triplexstructures can be formed from strand invasion of a duplex DNA with twoPNA strands. In this complex, the PNA/DNA/PNA triple helix portion andthe PNA/DNA duplex portion both produce displacement of thepyrimidine-rich triple helix, creating an altered structure that hasbeen shown to strongly provoke the nucleotide excision repair pathwayand to activate the site for recombination with the donoroligonucleotide. Two PNA strands can also be linked together to form abis-PNA molecule.

The triplex-forming molecules are useful to induce site-specifichomologous recombination in mammalian cells when used in combinationwith one or more donor oligonucleotides which provides the correctedsequence. Donor oligonucleotides can be tethered to triplex-formingmolecules or can be separate from the triplex-forming molecules. Thedonor oligonucleotides can contain at least one nucleotide mutation,insertion or deletion relative to the target duplex DNA.

Double duplex-forming molecules, such as a pair of pseudocomplementaryoligonucleotides, can also induce recombination with a donoroligonucleotide at a chromosomal site. Use of pseudocomplementaryoligonucleotides in targeted gene therapy is described in U.S. PublishedApplication No. 2011/0262406. Pseudocomplementary oligonucleotides arecomplementary oligonucleotides that contain one or more modificationssuch that they do not recognize or hybridize to each other, for exampledue to steric hindrance, but each can recognize and hybridize tocomplementary nucleic acid strands at the target site. In someembodiments, pseudocomplementary oligonucleotides are pseudocomplemenarypeptide nucleic acids (pcPNAs). Pseudocomplementary oligonucleotides canbe more efficient and provide increased target site flexibility overmethods of induced recombination such as triple-helix oligonucleotidesand bis-peptide nucleic acids which require a polypurine sequence in thetarget double-stranded DNA.

B. In Vitro Methods

The disclosed compositions can be used in a method of delivering activeagents to cells in vitro. For example, the nanolipogels can be used forin vitro transfection of cells. The method typically involves contactingthe cells with nanolipogels including a polynucleotide in an effectiveamount to introduce the polynucleotide into the cell's cytoplasm. Insome embodiments, the polynucleotide is delivered to the cells in aneffective amount to change the genotype or a phenotype of the cell. Thecells can be primary cells isolated from a subject, or cells of anestablished cell line. The cells can be of a homogenous cell type, orcan be a heterogeneous mixture of different cells types. For example,the polyplexes can be introduced into the cytoplasm of cells from aheterogenous cell line possessing cells of different types, such as in afeeder cell culture, or a mixed culture in various states ofdifferentiation. The cells can be a transformed cell line that can bemaintained indefinitely in cell culture. Exemplary cell lines are thoseavailable from American Type Culture Collection including tumor celllines.

Any eukaryotic cell can be transfected to produce cells that express aspecific nucleic acid, for example, a metabolic gene, including primarycells as well as established cell lines. Suitable types of cellsinclude, but are not limited to, undifferentiated or partiallydifferentiated cells including stem cells, totipotent cells, pluripotentcells, embryonic stem cells, inner mass cells, adult stem cells, bonemarrow cells, cells from umbilical cord blood, and cells derived fromectoderm, mesoderm, or endoderm. Suitable differentiated cells includesomatic cells, neuronal cells, skeletal muscle, smooth muscle,pancreatic cells, liver cells, and cardiac cells. In another embodiment,siRNA, antisense polynucleotides (including siRNA or antisensepolynucleotides) or inhibitory RNA can be transfected into a cell usingthe compositions described herein.

The methods are particularly useful in the field of personalizedtherapy, for example, to repair a defective gene, de-differentiatecells, or reprogram cells. For example, target cells are first isolatedfrom a donor using methods known in the art, contacted with thenanolipogel including a polynucleotide causing a change to the in vitro(ex vivo), and administered to a patient in need thereof. Sources orcells include cells harvested directly from the patient or anallographic donor. In preferred embodiments, the target cells to beadministered to a subject will be autologous, e.g. derived from thesubject, or syngenic. Allogeneic cells can also be isolated fromantigenically matched, genetically unrelated donors (identified througha national registry), or by using target cells obtained or derived froma genetically related sibling or parent.

Cells can be selected by positive and/or negative selection techniques.For example, antibodies binding a particular cell surface protein may beconjugated to magnetic beads and immunogenic procedures utilized torecover the desired cell type. It may be desirable to enrich the targetcells prior to transient transfection. As used herein in the context ofcompositions enriched for a particular target cell, “enriched” indicatesa proportion of a desirable element (e.g. the target cell) which ishigher than that found in the natural source of the cells. A compositionof cells may be enriched over a natural source of the cells by at leastone order of magnitude, preferably two or three orders, and morepreferably 10, 100, 200, or 1000 orders of magnitude. Once target cellshave been isolated, they may be propagated by growing in suitable mediumaccording to established methods known in the art. Established celllines may also be useful in for the methods. The cells can be storedfrozen before transfection, if necessary.

Next the cells are contacted with the disclosed composition in vitro torepair, de-differentiate, re-differentiate, and/or re-program the cell.The cells can be monitored, and the desired cell type can be selectedfor therapeutic administration. For examples, in some embodiments thedisclosed methods are be used to create allogeneic pluripotent ormultipotent cells (i.e., stem cells) from differentiated cells, or tochange the phenotype of immune cells.

Following repair, de-differentiation, and/or re-differentiation and/orreprogramming, the cells are administered to a patient in need thereof.In the most preferred embodiments, the cells are isolated from andadministered back to the same patient. In alternative embodiments, thecells are isolated from one patient, and administered to a secondpatient. The method can also be used to produce frozen stocks of alteredcells which can be stored long-term, for later use. In one embodiment,fibroblasts, keratinocytes or hematopoietic stem cells are isolated froma patient and repaired, de-differentiated, or reprogrammed in vitro toprovide therapeutic cells for the patient.

C. Diseases to be Treated

The compositions including nanolipogel delivery vehicles can be used totreat a variety of diseases and conditions, for example, cancer andinfectious diseases. The compositions can be administered to the subjecttherapeutically or prophylactically. Examplary therapeutic andprophylactic strategies are discussed in more detail below and in theExamples.

For example, in some embodiments, a cell penetrating peptide, also knownas cell permeable peptides, protein transduction domains (PTDs),membrane translocating sequences (MTSs) and Trojan peptides, (forexample a stuimulus-responsive cell penetrating peptide) is a conjugatedto a dendrimer in a nanolipogel formulation. Cell penetrating peptidesinclude, but are not limited to, virus-derived or mimicking polymerssuch as TAT, influenza fusion peptide, rabies virus glycoproteinfragment (RVG), neuropilin, penetratin, and polyarginines. Anaspec hascommercially available CPPs:

The nanolipogel can be used to deliver active agents to cells including,but not limited to difficult to penetrate cells, HIV infected cells, Tcell lymphomas, and B cells.

In some embodiments, the nanolipogel includes a death receptor agonist(such as Fas/CD95 ligand and TRAIL/Apo2L) and death receptors (such asFas/CD95, TRAIL-R1/DR4, and TRAIL-R2/DR5) which is involved inimmune-mediated neutralization of activated or autoreactive lymphocytes,virus-infected cells, and tumor cells. Dysregulation of deathreceptor-dependent apoptotic signaling pathways has been implicated inthe development of autoimmune diseases, immunodeficiency, and cancer.Moreover, the death ligand TRAIL has gained considerable interest as apotential anticancer agent, given its ability to induce apoptosis oftumor cells without affecting most types of untransformed cells. TheFLICE-inhibitory protein (FLIP) potently blocks TRAIL-mediated celldeath by interfering with caspase-8 activation. Pharmacologicdown-regulation of FLIP might serve as a therapeutic means to sensitizetumor cells to apoptosis induction by TRAIL. Accordingly, death ligandsor receptors can be incorporated onto or into the nanolipogel as thetargeting moiety and/or as the active agent to enhance cell specificdelivery and to sensitivity target cells, such as a cancer cells orvirally transformed cells, to apoptosis.

In some embodiments, the nanolipogel includes a moiety that specificallytargets a sirtuin. Sirtuin or Sir2 proteins are a class of proteins thatpossess either histone deacetylase or mono-ribosyltransferase activity.Sirtuins regulate important biological pathways in bacteria, archaea andeukaryotes, and have been implicated in influencing aging and regulatingtranscription, apoptosis and stress resistance, as well as energyefficiency and alertness during low-calorie situations. Accordingly,Sirtuin or Sir2 proteins can be targeted as part of an anti-agingprophylactic or therapeutic strategy.

In some embodiments, the active agent(s) includes a Histone deacetylaseinhibitor (HDACi). HDACi are chemical compounds that interfere with thefunction of HDAC enzymes. They inhibit HDAC enzyme activity, andtherefore, tip the equilibrium in favor of acetylated histones. HDACican also affect the activity of many non-histone proteins, as they canalso be targeted by lysine acetylation/deacetylation, leading to anincreased episode of acetylation of many gene clusters, leading to anincrease of transcription activity and, subsequently, upregulation ofspecific genes. In some embodiments, the active agent includes achemotherapeutic agent. Co-delivery of an HDACi and a chemotherapeuticdrug may be particularly effective for treating cancers, includingmulti-drug resistant cancers such as pancreatic cancer and melanoma.

In some embodiments, the nanolipogel is part of a vaccine strategy. Forexample, the nanolipogel can be used to deliver an antigen, animmunostimulant, an adjuvant, or a combination thereof. In someembodiments, the nanolipogel includes a target moiety that directs thedelivery vehicle to specific immune cells, for example, antigenpresenting cells such as dendritic cells. In some embodiments, thenanolipogel includes one or more antigen presenting cell targetingmoieties displayed on the outer shell, and TLR ligands inside or outsidethe nanolipogel, alone or in combination with an antigen. The antigencan be any known antigen, for example, an antigen derived from abacteria, a virus, a fungi, a parasite, or another microbe, or tumorantigens or environmental antigens.

In some embodiments the nanolipogel includes pH responsive elements sothat the contents of the nanolipogel are released upon encountering alow pH. This strategy can be employed to increase delivery of thenanolipogel contents to tumor cells or microenvironments or cardiaccells under hypoxic conditions. In some embodiments, the nanolipogelsare used in photodynamic therapy. For example, pH-sensitive nanolipogelcan release a photodynamic therapy agent such as hypercirin, the area oftreatment, for example tumor cells, or a tumor microenvironment.

In some embodiments, the active agent(s) includes a TranscriptionActivator-Like Effector Nucleases (TALENs). TALENs are artificialrestriction enzymes generated by fusing the TAL effector DNA bindingdomain to a DNA cleavage domain. TALENs can be employed for efficient,programmable, and specific DNA cleavage and represent powerful tools forgenome editing in situ. Synthetic transcription factors using TALEdomain constructs can also be used for gene regulation by pairing theTALE DNA binding domain with an endogenous activation domain affectingexpression at specific sites in complex genomes. Transcriptionactivator-like effectors (TALEs) can be quickly engineered to bindpractically any DNA sequence. Accordingly, TALENs can be used in genetherapy methods, for example to edit HIV-associated genes such as CCR5,or to treat monogenetic mutations in genetic diseases such as cysticfibrosis.

In some embodiments, the nanolipogel includes a pathogen-associatedmolecular pattern molecule (PAMP) targeting moiety. PAMPs are smallmolecular motifs associated with groups of pathogens, that arerecognized by cells of the innate immune system. They are recognized byToll-like receptors (TLRs) and other pattern recognition receptors(PRRs) in both plants and animals. They activate innate immuneresponses, protecting the host from infection, by identifying someconserved non-self molecules. For example, bacterial Lipopolysaccharide(LPS), an endotoxin found on the bacterial cell membrane of a bacterium,is considered to be the prototypical PAMP. LPS is specificallyrecognized by TLR 4, a recognition receptor of the innate immune system.Other PAMPs include, but are not limited to, bacterial flagellin(recognized by TLR 5), lipoteichoic acid from Gram positive bacteria,peptidoglycan, and nucleic acid variants normally associated withviruses, such as double-stranded RNA (dsRNA), recognized by TLR 3 orunmethylated CpG motifs, recognized by TLR 9. Accordingly, one or morePAMPs can be used to increase an immune response against an infectiousdisease.

In some embodiments, the nanolipogel includes a damage-associatedmolecular pattern molecule (DAMP). DAMPs include intracellular moleculesreleased by activated or necrotic cells and extracellular matrixmolecules that are upregulated upon injury or degraded following tissuedamage. DAMPs are danger signals that alert the immune signal to tissuedamage upon infection and tissue damage, but have also been implicatedin excessive inflammation including rheumatoid arthritis, cancer, andatherosclerosis (Piccinini and Midwood, Mediators of Inflammation, Vol.2010, Article ID 672395, 21 pages.). For example, in some embodiments,DAMPs can be used as part of a nanolipogel strategy to induce an immuneresponse wherein the DAMPs mimic a necrotic cell. Exemplary DAMPsinclude, but are not limited to, F-actin, HMGB1 (high mobility group boxprotein-1), S100A8/S00A9, heat-shock proteins, uric acid and DNA. Insome embodiments, DAMPs are incorporated into a vaccine strategy, forexample, a cancer vaccine strategy. For example, DAMP can be deliveredto immune cells using a nanolipogel decorated with an antigen presentingcell targeting ligand. In some embodiments, the cancer vaccine strategyalso include delivery of one or more tumor associated antigens.

D. Exemplary Disease Treatment Strategies

1. Methods of Immunotherapy and Cancer Treatment

One mechanism behind how melanomas and other cancers evade the antitumorresponse is postulated to be the inability of the innate immune systemto recognize the tumor as ‘non-self’. This may occur due to thesecretion of a number of immunosuppressive factors by tumor cells,including transforming growth factor-β (TGF-β), a pleiotropic cytokinethat decreases natural killer cell (NK) number and function andcytotoxic T lymphocyte (CTL) function while increasing the number ofregulatory T lymphocytes (Tregs). TGF-β activity has been extensivelyevaluated in a number of animal disease systems, including murine tumormodels, and its secretion is suspected to thwart high-dose interleukin-2(IL-2) therapy, which is supposed to enhance NK and CTL activity againstmelanomas and renal cell cancers but lacks efficacy in the majority ofpatients. This has led groups to evaluate strategies to counteractimmunosuppressive factors secreted from tumors, including TGF-β.Although the exact source of intratumoral TGF-β has not beenwell-established, the cytokine has been found at high levels in a largenumber of different tumors, including melanomas. It is believed thatTGF-β is pivotal for tumor cell growth and differentiation as well asmaintaining an immunosuppressive environment to protect an establishedtumor from the host immune response, rendering it an ideal target forcancer therapies. In particular, its suppressive effect on the number ofNK cells present in tumor beds may be crucial for immune tolerance, asthese cells play an important role in the anti-tumor response.

Examples demonstrate the efficacy of simultaneous, sustained release ofIL-2 and SB against tumors. Given the vastly different physiochemicalproperties of IL-2, a soluble 17 kDa protein, and SB, a smallhydrophobic drug (Log P=4.33), co-encapsulation for sustained release ofboth agents presented a challenge for conventional particletechnologies. For example, liposomes are easily modified forencapsulation of small hydrophilic molecules, and even proteins, but thestability of these formulations and the release profiles of encapsulatedagents are not easily controlled. Biodegradable solid particles, on theother hand, such as those fabricated from poly(lactic-co-glycolic acid)(PLGA), are highly stable and have controllable release characteristics,but pose complications for facile encapsulation and controlled releaseof therapeutic cytokines or for combinatorial delivery.

Metastatic melanoma is highly aggressive, leaving untreated patientswith a median survival of less than 12 months. The ineffectiveness ofsurgical interventions, radiation and cytotoxic chemotherapies hasresulted in immunotherapy as the primary treatment modality.Approximately 5% of patients with metastatic melanoma achieve durablecomplete remissions when treated with high dose IL-2, presumably viainduction or expansion of activation of melanoma-specific T cellresponses. However, because high dose-related toxicity of IL-2 hampersits therapeutic benefits, newer generation formulations aim to reducethe administered dose by increasing the half-life of the cytokine incirculation. Some examples include fusion proteins (IL-2/Ig), pegylatedIL-2, IL-2/anti-IL-2 complexes, liposomal formulations and viral andplasmid vectors. Nanolipogel combination delivery of TGF-β inhibitor andIL-2 enhances tumor immunotherapy. The sustained release of two diversechemical agents from nanolipogels elicits an impressive anti-tumoreffect. The tumor microenvironment thwarts conventional imnunotherapythrough multiple immunologic mechanisms. Several of these mechanisms arebelieved to include secretion of the transforming growth factor-β(TGF-β), which stunts local tumor immune responses. Thus, even a highdose of interleukin-2 (IL-2), a conventional cytokine FDA-approvedtreatment for metastatic melanoma, only induces limited responses.

To overcome the immunoinhibitory nature of the tumor microenvironment, avehicle is needed for releasing to the tumor microenvironment in acontrolled fashion an inhibitor of TGF-β together with IL-2. Onewell-known inhibitor of TGF-β is2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride (known as SB-505124 and referred to herein as SB).However, given the vastly different physiochemical properties of IL-2, asoluble 17 kDa protein, and SB, a small hydrophobic drug (Log P=4.33),co-encapsulation for sustained release of both agents presented achallenge for conventional particle technologies. The nanolipogelplatform combines features of both liposomes and polymer-based particlesfor encapsulation and sustained delivery of therapeutic proteins andsmall molecule hydrophobic drugs.

The sustained delivery of both IL-2 and SB from this system inducespotent antitumor immune responses in a B16/B6 mouse model of melanomaafter intratumoral or systemic administration. Nanolipogels releasingTGF-β inhibitor and IL-2 significantly delayed tumor growth, increasedsurvival of tumor-bearing mice and increased intratumoral natural killercells (NK). Additionally, induction of remission with this combinationtherapy was mediated through activation of both the innate and adaptivearms of the immune response, with the innate arm playing a critical rolein mediating antitumor activity in vivo. The formulation offers anadvantage by not only increasing the cytokine half-life in circulationbut also by co-delivering in a sustained manner SB, a potent pleiotropicinhibitor that suppresses the tumor's ability to thwart an immuneresponse.

Combination therapies that stimulate immune responses while overcomingthe tumor inhibitory environment are attractive modalities for cancerimmunotherapy. The nanolipogel platform which has been demonstrated tobe effective in combination therapy in the Examples below in turnprovide a platform for therapy in a variety of diseases in which it isdesirable to simultaneously utilize combinations of drugs in a treatmentregimen. Such combination therapies, and the diseases in which they areuseful, are well known in the art.

Exemplary cancer therapies are outlined in Table 1 below. The tablepresents a pathological aberrance that is addressed by the therapy; thecells target(s) of the therapy; one, two, or three therapeutic moleculesthat can be delivered by the nanolipogels alone or in any combinationthereof; a desired target or targeting moiety can be used to target thenanoliposomes; the preferred delivery mechanism; and intended effects ofthe therapy.

TABLE 1 Exemplary Cancer Therapies and Strategies Delivered PathologicalCell Molecule Delivered Targeting Delivery Disease Abberrance Target (s)(DM) 1: Molecule 2: DM3: Strategy Mechanism Effect Multi-Drug chemodrugs kill cancer cells histone chemo drug cancer- preferrably HDACiResistant rapidly dividing deacetylase dependent HDACi is initiates(MDR) cancer cells cancer inhibitor EPR, folate, delivered firsttranscription, senescence (HDACi) TAA, etc resensitizes evades drugscells to chemo MDR Cancer induced Tregs iTregs siRNA against HDACi toiTreg (iTregs) FoxP3 modify surface enhance cancer epigenetics markersimmunoediting not nTreg MDR Cancer IL-10 Tregs IL-10 Tregs unclear. IL-2have same plays a suppressive role... fxn as FoxP3 iTregs in mouseCancer cancer cells tumor cells pHLIP- ATP, pH-sensitive other DAMPstumor cells create calreticulin HMGB1 could be mimic invisibilityemployed immunogenic to immune cell cells death, create inflammatoryresponse Cancer tumor-associated TAMs macrophages (TAMs) promotingdisease Unresectable BC-819 Pancreatic plasmid Cancer Unresectable, drugPancreatic HDAC chemo drug antibody for HDACi initiates MDR deliveryCancer Cells inhibitor tumor transcription, Pancreatic challenges,(SAHA, associated resensitizes Cancer resististance TSA, etc) antigencells to chemo (TAA) to chemo

2. Infectious Diseases

Non-limiting examples of additional diseases that can be treated usingthe compositions and methods disclosed herein include infectiousdiseases, viral or microbial, in which a combination antiviral orantibiotic regimen, respectively, is the desirable strategy. Forexample, an anti-HIV formulation could include activators to initiateHIV replication, inhibitors that prevent HIV infection of new cells anda mixture of death-inducers that are exclusively activated within theinfected cell with no harm befalling the others. The outer lipid shellcan be fabricated with an antibody that attaches specifically to amolecule expressed on all human T-cells. This serves as the targetingvehicle that protects the encased components and fuses with targetT-cells. The nanolipogel core is fabricated from a safe, FDA approvedpolymer encapsulating a ‘dendrimer’.

This inner dendrimer core is complexed with 1) A HDAC inhibitor (HDACi)that activates HIV. This agent inhibits enzymes known as histonedeacetylases (HDACs) that constantly remove acetyl groups on histones,enabling continued binding of histones to chromosomal DNA, which helpskeep HIV latent; 2) A plasmid that encodes RNA-inhibitors called shortinterfering RNA (siRNA) that bind exclusively to the viral RNA anddestroy it by a cellular pathway called RNA interference. siRNAs can bedesigned to target only the intended mRNA target with minimal sideeffects providing an immense advantage. These siRNAs can be expressed inall T-cells so as to prevent viral spread from the infected cell as wellas productive infection in uninfected cells; and 3) Another plasmid thatencodes siRNAs controlled by a promoter that is activated exclusively bythe HIV proteins tat and rev and hence expressed only in infected cells.These siRNAs are designed to bind and destroy RNAs of proteins thatpromote cell survival. Upon attachment the entire system is internalizedby the cellular machinery without altering cell physiology orhomeostasis, the outermost particle breaks down to release the innerdrug/gene-complexed core, which is further degraded releasing thecomponents. Thus the system is doubly regulated for attachment only toT-cells, the reservoirs for latent HIV and selective destruction ofinfected T-cell reservoirs.

Exemplary therapies and strategies of treating HIV and other infectiousdiseases are outlined in Table 2 below. The table presents apathological aberrance that is addressed by the therapy; the cellstarget(s) of the therapy; one, two, or three therapeutic molecules thatcan be delivered by the nanolipogels alone or in any combinationthereof; a desired target or targeting moiety can be used to target thenanoliposomes; the preferred delivery mechanism; and intended effects ofthe therapy.

TABLE 2 Exemplary HIV Therapies and Strategies Delivered PathologicalCell Molecule Delivered Targeting Delivery Disease Abberrance Target (s)(DM) 1: Molecule 2: DM3: Strategy Mechanism Effect HIV latently latentHDAC siRNA against anti-CD7- preferrably, expressed infected T infectedinhibitor specific HIV palmitate HDACi is proteins Cells evade CD4+(SAHA, proteins released first are toxic, HAART TSA, etc) necessary forlatent cells propogation die HIV Tregs attenuate FoxP3+ Tregs, siRNAtargeting anti-CD4 deactivate HIV immune- perhaps Ag- FoxP3 permissiveactivation, specific Tregs suppress immune response to HIV + otherpathogens HIV HAART drugs infected CD4+ HAART drug 1 HAART drug 2 HAARTdrug 3 anti-CD4 bundle HAART go T cells drugs everywhere to increaseefficiency, decrease side effects HIV CCR5 allows HIV CD4+ T cells ZincFinger anti-CD4 prophylactic protect against entry Nuclease (ZFN)strategy HIV or TALEN cellular entry against CCR5 HIV MHC-restrictedAPCs (DCs, ZFN or passive or modulate HIV epitopes macrophages) TALEN toTLR ligand, TCR/MHC- do not change anti- HIV epitope mount sufficientMHC DEC205 interactions immune response sequence to initiate robustimmune response HIV MHC-restricted cells that will optimal pH-responsivemake lots HIV epitopes interface with MHC-HIV insertion of of APC do notmount T cells epitope MHC/epitopes presenting sufficient even moleculesfor TCR HIV epitopes immune epithelial cells presentation in ways tresponse propogate immun response Epstein-Barr B cells infectedEBV-infected B siRNA against anti-B220, Ayman's siRNA prevent virusVirus with EBV, cells EBV targets others sequence from virus replicatesreplicating purge disease Malaria parasite infected RBCs plasmid DNAmore plasmid Choukri transform parasite, infects RBCs DNA Mamounidentify drug plasmids resistant infected RBCs Leishmania parasite livesin infected myriad small excess mannose, expel macrophages macrophagesmolecule drugs dendrimer to Fc parasite from help break fragment,endolysosome, endosome etc kill it

3. Other Indications

Yet another non-limiting example is cardiovascular disease in whichcombination therapies well known in the art are in use to simultaneouslylower both blood pressure and cholesterol levels. It is notable in anumber of such diseases that modulating biodistribution would also offeradvantages in the treatment strategy and as some of the Examples in thisinvention demonstrate, the use of nanolipogels offers promise withregard to control of biodistribution.

The success of combination therapies is a safe and flexible deliveryplatform that releases a variety of effector molecules with differentphysiochemical properties to tumor beds in a sustained fashion, such asthe nanolipogel delivery system developed in this work. Recent work hasdemonstrated that the combination of another TGF-β receptor-I inhibitor(LY364947) and the cytotoxic chemotherapeutic agent, doxorubicin, waseffective in a particulate formulation against pancreatic and gastriccarcinomas. Cytokines, including IL-2, represent a complex network ofsoluble proteins critical for immunological and effector cell function.In a similar fashion, small molecule hydrophobic drugs, such as TGF-βantagonists, represent a class of immunomodulators that can overcomebarriers posed by tumors to escape the immune response. The sustaineddelivery of these agents in combination to tumor beds can inducetherapeutic immune responses, while reducing the immune-resistant natureof the tumor microenvironment.

The B16 melanoma model was used to validate that the difficulties inensuring simultaneous, synergistic delivery of both labile proteins andsmall hydrophobic molecules can be addressed by rational engineering ofa nanoscale delivery system fabricated from inert, biodegradablecomponents with a history of use, individually, in different drugdelivery applications. The Examples illustrate that the activation ofthe innate arm of the immune system is a critical immunologic mechanismunderlying the synergistic effects of simultaneously delivering IL-2 andSB, resulting in delayed tumor growth and enhanced survival oftumor-bearing mice. Administration of SB in combination with IL-2stimulated the innate immune system, greatly increasing the number of NKin tumors in mice receiving this combination. Absence of therapeuticefficacy following NK depletion demonstrated that stimulation of theinnate arm by nanoparticles releasing both agents was crucial forachieving an improvement in survival in this model. Particles releasingSB, IL-2 alone or the combination also stimulated the adaptive immunesystem, enhancing activated CD8⁺:Treg ratios. These results show thatthe combination therapy can stimulate both arms of the immune systemsimultaneously.

Nanocarriers designed to release both soluble cytokines and hydrophobicdrug molecules in a sustained fashion, even without tumor targeting, canbe used to simultaneously deliver a combination of key immunomodulatorsto decrease the local immunosuppressive environment and enhanceantitumor responses. The combination of IL-2 and a TGF-β antagonist atthe tumor site led to significant tumor delay, and, in select cases,macroscopic remissions in the B16/B6 murine melanoma model. It isnotable that aggressive tumors such as melanoma inherently develop leakyvasculature with 100 to 800 nm pores due to rapid vessel formation tosustain a rapidly growing tumor. This defect in vasculature and poorlymphatic drainage results in enhanced permeation and retention ofnanoparticles within the tumor bed. This is often called the enhancedpermeation and retention (EPR) and is a form of ‘passive targeting.’ Thebasis for increased accumulation of drug-loaded nanoparticles in tumorsover normal tissues is that, unlike tumor beds supplied by leakyvasculature, normal tissues contain capillaries with tight junctionsthat are less permeable to nanosized particles. Passive targeting cantherefore result in several fold increases in particulate concentrationsin solid tumors compared to free administration of antibodies or otherdrugs and may explain the increased survival and effective treatment ofmetastasis observed after intravenous injection.

This is one example of how nanolipogels can be used to advantage withregard to biodistribution. Other strategies to further increase thissurvival index include increasing the frequency of injections, dosageper injection or inclusion of tumor retention ligands on the surface ofthe nanoparticle to improve the selective delivery of agents andretention in the tumor microenvironment. The activity of tumorinfilitrating lymphocytes and NK can be enhanced by the delivery ofadditional cytokines such as IL-15, which belongs to the IL-2 family ofcytokines and which can function to enhance the survival of IL-2activated cells.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1: Preparation of Nanolipogels for Delivery ofAnti-Tumor Molecules

Materials and Methods

Nanolipogel Synthesis.

“Nanolipogel” (“nLG”) particles were fabricated from a degradablepolymer (FIG. 1B). Liposomes were used as nanoscale molds forphoto-initiated hydrogel formation. To achieve sustained release of thehydrophobic drug in conjunction with encapsulated proteins,methacrylate-conjugated β-cyclodextrins (CDs) were incorporated into theinterior of the liposomes. β-cyclodextrins have a long history assolublization agents for hydrophobic compounds and are key excipients invarious pharmaceutical formulations. This formulation procedure enabledco-encapsulation of both proteins as well as small hydrophobic drugswithin the interior of the lipid bilayer (FIG. 1A-1B).

Conjugated CDs were created by reaction of succinylated-CDs withphotosensitive methacrylate groups through hydrolysable ester groups.(FIG. 1A) Complexation of SB or rhodamine (for imaging) withfunctionalized CD was verified by proton nuclear magnetic resonance (¹HNMR) on a 500 MHz Bruker spectrometer. All samples were dissolved in1-10 mg/ml in D₂O for characterization with the solvent as a reference.

PLA-PEG-PLA diacrylate was synthesized in two steps according toSawhney, et al. Macromole 26, 581-587 (1993). All chemicals werepurchased from Sigma unless otherwise noted and were of ACS grade orhigher. α,ω-dihydroxy poly(ethylene oxide) with a molecular weight of4000 g/mol, 3,6-dimethyl-1,4-dioxane-2,5-dione (dl-lactide), and tin(II)2-ethylhexanoate (stannous octoate) were charged into a round-bottomflask under nitrogen in a 5:1:0.0075 mol ratio and the reaction wasstirred under vacuum at 200° C. for 4 hours, followed by stirring at160° C. for 2 hours. After cooling to room temperature, the resultingcopolymer was dissolved in dichloromethane and precipitated in anhydrousether. This intermediate was dissolved in dichloromethane (10 g/mL) andcooled to 0° C. in an ice bath. Per 10 g of polymer intermediate, 440 μLtriethylamine and 530 μL acryloyl chloride were added under nitrogen andthe reaction mixture was stirred for 12 hours at 0° C. and 12 hours atroom temperature. The mixture was filtered and the resulting polymer wasprecipitated in diethyl ether. The final polymer was redissolved indichloromethane, re-precipitated in hexanes and characterized by FTIRand NMR.

The complexation of Rhodamine and SB505124 with cyclodextrins wasexamined by proton nuclear magnetic resonance (1H NMR) spectroscopy on a500 MHz Bruker spectrometer.

Nanolipogel Formulation.

All lipids were obtained from Avanti Polar Lipids and used withoutfurther preparation. Phosphatidyl choline (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol were mixed in chloroform in a3:1:1 molar ratio and liposomes were formulated using a remote loadingtechnique of Peer, et al. Science 319, 627-630 (2008). Lipid-labeledfluorescent liposomes were formulated by incorporation of 10%1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)2000-N′-carboxyfluorescein] (DSPE-PEG-Fluorescein). Briefly, thedissolved lipids were mixed in a glass scintillation vial, followed bycomplete solvent removal with a directed nitrogen stream. This formed athin lipid film on the inner glass surfaces, which was rehydrated by theaddition of 1× phosphate buffered saline (PBS). Cycles of thirty secondvortexing followed by 5 min idle sitting at room temperature wererepeated ten times and the resulting multilamellar liposomes wereextruded 10 times through a 5 m polycarbonate membrane (Whatman), 10times through a 1 μm membrane and finally 11 times through a 100 nmusing a LIPEX extruder (Northern Lipids, Inc.). The resultingunilamellar liposomes were then frozen and lyophilized.

Lyophilized liposomes were reconstituted with a solution containing 5%(w/v) polymer (FIG. 1B) and 2.5 mg/mL Ciba Irgacure 2959 as thephotoinitiator and: no other additive (nLG-Empty), 9 mg f-CD-solubilizedSB/100 mg nLG (nLG-SB; SB505124, Sigma), 1 μg IL-2/100 mg lipids(LG-IL-2; Aldesleukin Proleukin, Novartis), or both f-CD-solubilized SBand IL-2 (nLG-SB+IL-2). CD (randomly succinylated β-CD; CTD, Inc.) wasfunctionalized with 2-aminoethyl methacrylate by stirring a 1:3 molarratio of the compounds in 1×PBS for 1 hour at room temperature. SB wasincorporated into f-CD by adding the drug dissolved in methanol to thef-CD. After 20 minutes of vigorous stirring at room temperature to formthe complexes, the methanol was evaporated with a directed stream ofnitrogen. The reconstitution step proceeded with 30 minutes of vortexingto rehydrate the liposomes. The liposomes were then irradiated under UVlight for 8 minutes with a Blak-Ray long wave ultraviolet lamp (Model B100) at a 10 cm working distance. Directly prior to UV irradiation, thesamples were diluted fivefold to prevent macroscale gellation. Theresulting nanolipogels were pelleted by centrifugation (five minutes at7200 ref) and resuspended in 1×PBS. This centrifugation/resuspensionprocedure was repeated three times. Nanolipogels were aliquotted andfrozen at −20° C. until further use. For consistency, all nanolipogelswere frozen prior to use (in vitro or in vivo). Final size anddispersity was confirmed by resuspending nanolipogels in 1×PBS foranalysis on a ZetaPALS dynamic light scattering instrument. The zetapotential of PC/cholesterol liposomes, PC/cholesterol/PE-PEG-NH₂liposomes, and nanolipogels were evaluated in 0.1×PBS using a Malvernnanosizer.

For TEM analysis, nanolipogel samples were stained with osmium tetroxideand then imaged on an FEI Tenai Biotwin microscope. Lipid-specificosmium tetroxide staining of cryosectioned samples had a localizedstaining pattern confined to the exterior membrane of the particle.

Results

Liposomes were used as nanoscale molds for photo-initiated hydrogelformation. To achieve sustained release of the hydrophobic drug inconjunction with encapsulated proteins, methacrylate-conjugatedβ-cyclodextrins (CDs) were incorporated into the interior of theliposomes. β-cyclodextrins have a long history as solublization agentsfor hydrophobic compounds and are key excipients in variouspharmaceutical formulations.

Complexation of SB or rhodamine (for imaging) with functionalized CD wasverified using ¹H NMR. The functionalized CD (f-CD) becomes covalentlybound to the liposome-encapsulated polymer matrix during photo-inducedpolymerization, thus the SB can only be released upon f-CD/SB hydrolysisof the polymer ester groups and subsequent diffusion out of thenanolipogel, enabling sustained release compared to the burst-dominatedrelease of SB in the absence of gelled CD. This system enabled controlover the release of remotely loaded IL-2 without compromising itsbioactivity and enabled simultaneous release of both protein and drugcompared to single component release. The release profile ofSB/IL-2-loaded nanolipogels was not altered by incubation in serum andrelease was substantively completed by 7 days.

To demonstrate the impact of polymerization in the nLG on the releaseprofile of SB and IL-2, release kinetics of both agents were comparedwith release from liposomes and solid poly(lactide-co-glycolide)nanoparticles (PLGA NPs) encapsulating both agents. Incorporation ofphotocured polymer in the nanolipogel vehicle enabled a more sustainedrelease of SB compared to liposomes and a more complete release comparedto conventional 50:50 (PLGA NPs) of the same diameter. The releasekinetics of the drug is seen to be intermediate between that ofdiffusion dependent release from liposomes and hydrolysis dependentrelease from PLGA. Comparative cumulative release of IL-2 fromliposomes, nanolipogels, and PLGA NPs demonstrated that encapsulation ofIL-2 in nanolipogels enabled better sustained release of cytokine.

The bioactivity of the SB and IL-2 were unaffected by lipogelincorporation. Encapsulation of IL-2 (80%) and/or drug (36%) did notsignificantly affect nanolipogel diameter; dynamic light scatteringanalysis revealed a mean diameter of 120 nm and polydispersity index of0.2. Liposomes and nanolipogels incorporating amine-terminated PEGylatedphosphatidyl ethanolamine demonstrated a neutral zeta potential,compared to the approximately −22±10 mV zeta potential of liposomesformulated with only phosphatidyl choline and cholesterol. Cryo-TEM ofnanolipogels showed the formation of spherical liposomal structures,detectable by light scattering even after disruption of the liposomalexterior by detergent, validating an inner gel core with approximatelythe same diameter as the intact nanolipogel. The in vitro cytotoxicityof this system was negligible.

To investigate the biodistribution and clearance of this platform,CD-solubilized rhodamine was used as a fluorescent surrogate markermodel for SB; rhodamine complexation with CD had been previously used toqualify guest-host interactions with CDs. This was confirmed here by ¹HNMR.

Encapsulation of SB or SB+IL-2 had no significant effect on particlemean diameter or polydispersity. FIG. 1D shows that the zeta potentialof liposomes and nanolipogels incorporating amine-terminated PE-PEG wasfound to be close to neutral. FIG. 1E shows the composition andformulation properties of the nanolipogel formulation. FIG. 1F shows thepolymer structure verified by ¹H NMR. Cryo-TEM of nanolipogelsdemonstrated the formation of spherical liposomal structures. FIG. 1Gshows that the photopolymerized polymer/CD forms nanoparticulatehydrogel structures that are detectable by light scattering even afterdisruption of the liposomal exterior by detergent.

Example 2: In Vitro Release and Bioactivity Studies

Materials and Methods

Controlled Release Studies.

To demonstrate the advantage of nanolipogel vehicles for controlledrelease of encapsulated agents over prolonged periods of time, a seriesof studies were conducted to evaluate in vitro release of nanolipogelparticles containing SB and/or IL-2. Release studies were performed at37° C. with constant agitation in 1×PBS+10% fetal bovine serum. At eachtime point the complete volume was removed and replaced with freshbuffer after centrifugation (five minutes at 7200 rcf). Nanolipogelswere resuspended by manual pipetting. Absorbance measurements todetermine SB concentrations were performed with a Beckman Coulter platereader at 300 nm. Absorbance readings from nLG-Empty particles weresubtracted from those obtained from nLG-SB particles to ensure readingswere due only to encapsulated SB. IL-2 release was determined using anIL-2 ELISA kit (BD Biosciences) with humanized capture (BD, 555051) andbiotinylated-detection (BD, 555040) antibodies according to themanufacturer's instructions. For IL-2 used in these studies, theinternational unit conversion was 22 MU=1.3 mg.

The functionalized CD (f-CD) becomes covalently bound to theliposome-encapsulated polymer matrix during photo-inducedpolymerization, thus the SB can only be released upon f-CD/SB hydrolysisof the polymer ester groups and subsequent diffusion out of thenanolipogel, enabling sustained release compared to the burst-dominatedrelease of SB in the absence of gelled CD.

Bioactivity Studies.

Cumulative release of nLG-IL-2 was performed at 1, 3, 5, and 7 days incomplete media [RPMI media (Gibco) with 10% fetal bovine serum (AtlantaBiological) and penicillin/streptomycin (Sigma) supplemented withL-glutamine (Sigma), non-essential amino acids (Gibco), Hepes buffer(Sigma), gentamicin (Sigma), and β-mercaptoethanol (Sigma)]. Splenocyteswere isolated from a B6 mouse and 1×10⁶ cells were added in 500 μL Tcell media to each well of a 24 well plate previously coated with 10μg/mL anti-CD3 (coated overnight in 1×PBS at 4° C.) and 5 μg/mL solubleanti-CD28 (BD Biosciences). The media from release studies was filteredthrough a 0.22-μm syringe filter (Whatman) and 500 μL was added to thewells. Additionally all wells contained 5 μg/mL, soluble anti-CD28 (BDBiosciences). Soluble IL-2 was added at varying concentrations tocontrol wells to as a standard. Cells were incubated at 37° C. andcellular stimulation was assessed after 72 hours using an IFN-γ ELISA(BD Biosciences).

Results

FIGS. 2A-2E are comparative release profiles from nLG, lipsomes andsolid polymer nanoparticles (PLGA). Cumulative CD- or methacrylatefunctionalized-CD (f-CD)-solubilized SB released from nLGs normalized byinitial carrier mass demonstrated that polymerization of nanolipogelsimproved the sustained nature of SB release (FIG. 2A). Hydroxypropylβ-CD was used for SB complexation with the unfunctionalized CD.Cumulative IL-2 released determined by ELISA (immunoactive) and by abioactivity study (bioactive) from nLGs normalized by initialnanolipogel mass demonstrated that bioactivity of IL-2 was unaffected byencapsulation (FIG. 2B). Release of SB and IL-2 was not affected byincubation of 10 mg nLG in 1 ml full serum (FIG. 2C). Comparativecumulative release of SB from liposomes, nanolipogels, and degradablepolymeric (poly lactide-co-glycolide) nanoparticles (PLGA NPs)demonstrated that incorporation of photo-cured polymer in thenanolipogel vehicle enabled better sustained release and more completerelease of cyclodextrin-solubilized SB (FIG. 2D). PLGA NPs (meandiameter=−150±50 nm) were prepared by using a modified water/oil/waterdouble emulsion technique. Liposomes were prepared in an identicalmanner as the nLG without the polymer core. Liposomes were loaded withIL-2 and SB similar to nanolipogels. The diminished percent ofencapsulated SB released from PLGA NPs is attributed to the interactionof the relatively hydrophobic polymer with SB. All particulateformulations were dissolved in 0.1N NaOH+1% SDS to determine 100%release at 7 days (arrow) (FIG. 2D). Comparative cumulative release ofIL-2 from liposomes, nanolipogels, and PLGA NPs demonstrated thatencapsulation of IL-2 in nanolipogels enabled better sustained releaseof cytokine. Cumulative release is presented as % of total IL-2 releasedthrough 7 days. (FIG. 2E) Data in all graphs represent mean oftriplicate samples ±1 standard deviation. FIG. 2F compares the sizes andloading of IL-2 and SB in PLGA, nanolipogels and liposomes.

This system enabled control over the release of remotely loaded IL-2without compromising its bioactivity. Loading of IL-2 in the polymerhydrogel space outside of the CD enabled simultaneous release of bothprotein and drug. The decreased total release of both components (FIG.2C) compared to single component release was likely due to stericlimitations within the interior of the nanolipogel or decreased loadingefficiency of SB and IL-2. The release profile of SB/IL-2-loadednanolipogels was not altered by incubation in serum and release wassubstantively completed by 7 days.

To demonstrate the impact of polymerization in the nanolipogel on therelease profile of SB and IL-2 the release kinetics of both agents werecompared with release from liposomes and solidpoly(lactide-co-glycolide) nanoparticles (PLGA NPs) encapsulating bothagents. Incorporation of photocured polymer in the nanolipogel vehicleenabled a more sustained release of SB compared to liposomes and a morecomplete release compared to conventional 50:50 (PLGA NPs) of the samediameter. The release kinetics of the drug is intermediate between thatof diffusion-dependent release from liposomes and hydrolysis-dependentrelease from PLGA. Comparative cumulative release of IL-2 fromliposomes, nanolipogels, and PLGA NPs demonstrated that encapsulation ofIL-2 in nanolipogels enabled better sustained release of cytokine.

Bioactivity.

Nanolipogel vehicles provide the wherewithal to control release ofencapsulated agents without compromising bioactivity. The bioactivity ofthe SB and IL-2 were unaffected by lipogel incorporation. IFN-γproduction was correlated with IL-2 concentration to determinebioactivity.

Example 3: Characterization of Nanolipogels

Encapsulation of IL-2 (80%) and/or drug (36%) did not significantlyaffect nanolipogel diameter; dynamic light scattering analysis revealeda mean diameter of 120 nm and polydispersity index of 0.2. Liposomes andnanolipogels incorporating amine-terminated PEGylated phosphatidylethanolamine demonstrated a neutral zeta potential, compared to the−22110 mV zeta potential of liposomes formulated with only phosphatidylcholine and cholesterol. Cryo-TEM of nanolipogels showed the formationof spherical liposomal structures, detectable by light scattering evenafter disruption of the liposomal exterior by detergent, validating aninner gel core with approximately the same diameter as the intactnanolipogel. The in vitro cytotoxicity of this system was negligible.

Example 4: Biodistribution

To investigate the biodistribution and clearance of the nanolipogels,CD-solubilized rhodamine was as a fluorescent surrogate marker model forSB; rhodamine complexation with CD had been previously used to qualifyguest-host interactions with CDs. This was confirmed by ¹H NMR. The invivo pharmacokinetics of rhodamine following systemic administration wasevaluated in healthy mice receiving a single intravenous administrationof nLG-rhod, an equivalent dose of free rhodamine, or PBS control viatail vein injection.

Results

Spectrofluorometric analysis of rhodamine extracted from blood showed15.7±4.1% and 7.7±3.7% (mean±s.d.) of the initial dose of nanolipogelremaining at 1 and 24 hours respectively post-injection. Free rhodaminewas rapidly cleared and was not detectable in blood at any of the timepoints taken following injection.

FIGS. 3A-3G are graphs showing controlled release, clearance, andbiodistribution. The distribution of both nanolipogel carrier andencapsulated drug payload was investigated using dual-labeled NLG;fluorescein-labeled phosphoethanolamine was incorporated into the lipidcomponent of rhodamine-loaded nanolipogels. Spectrofluorimetric analysisat 540/625 nm and 490/517 nm showed dose-dependent fluorescence with nospectral overlap. FIG. 3A is a graph of cumulative IL-2 (ng/mg nLG) anddrug (μg SB/mg nLG) released from co-loaded nLGs normalized by carriermass. Error bars in all plots represent ±1 standard deviation. FIG. 3Bis a graph showing clearance (percent of initial dose) of drug dose overtime in days: Encapsulation in nanolipogels significantly increased theremaining percentage of initial dose in the blood at 1 and 24 hourspost-injection (two population t test, p<0.01 ###). FIG. 3C is a graphof whole body distribution. Mice received a single dose ofrhodamine-loaded nanolipogel or soluble rhodamine (in saline) viaintravenous tail vein injection. Animals were sacrificed at 1, 24, 48,and 72 hours post-injection for extraction and quantification offluorescence.

Whole body biodistribution was determined with rhodamine labeling.Significantly higher (two population t test, p<0.01) amounts ofrhodamine were detected in the major organs of nanolipogel-treatedanimals compared to animals injected with free dye. FIG. 3D is a graphof time dependent accumulation n in subcutaneous tumor: Cumulativerhodamine tumor penetration (circles) after B16 peritumoral injection inB6 mice. Peritumoral tissue was collected to quantify the remaining doseof nLG surrounding the tumor (squares). Controlled release demonstratesrelease of rhodamine, but not lipid (FIG. 3E). Mice bearing subcutaneousB16 tumors received a single IV (tail vein) injection of dual-labeledNLG. Animals were sacrificed at 1, 2, 3, and 7 days post injection andtissues collected for homogenization, extraction, and quantification ofrhodamine and fluorescein-PE. Analysis of serum showed prolongedcirculation of both encapsulant and delivery vehicle. Similar patternsof biodistribution were observed between lipid (FIG. 3F) and drugpayload (FIG. 3G), with highest accumulations of drug occurring in thelungs and liver.

Analysis of the biodistribution to major organs showed that the lungs,liver and kidney were primary sites of accumulation of bothnanolipogel-encapsulated rhodamine and free rhodamine. Encapsulation innanolipogel increased both the total initial dose to most tissues aswell as the cumulative dose over three days.

Example 5: Cytotoxic and Safety Studies

Materials and Methods

Cell titer blue (Invitrogen) was used as a cell viability markeraccording to the manufacturer's instructions. Chinese Hamster Ovary(CHO) cells (ATCC) were placed in 96 well plates at a density of 5×10⁴cells/well (except the standard, which contained a serial dilution ofthe number of cells). Cells were incubated for 24 hours at 37° C. withserial dilutions of IX PBS (positive control), sodium azide (negativecontrol; Sigma), liposomes, or nanolipogels. Liposomes were fabricatedsimilarly to nanolipogels but, after lyophilization, were reconstitutedwith pure 1×PBS and were not subjected to UV irradiation. Nanolipogelswere from the nLG-Empty group. After 24 hours the cell titer bluereagent was added (20 μL/100 μL volume). The cells were furtherincubated for 4 hours at 37° C., after which they were pelleted and thefluorescence of the supernatant was measured. 100% cell survival isdefined as the average of survival from the IX PBS group and 0% survivalthat from the azide group. All samples were run in triplicate and theexperiment was repeated three times with similar results.

To examine the in vivo safety of nanolipogel particles, C57/B16 micewere administered a single intravenous dose of nanolipogels and acutetoxicology was measured 7 days later. Lung toxicity was evaluated byhistology to determine if systemically administered nanolipogels inducedany acute inflammation.

Results

No statistically significant toxic effects were observed from theadministration of empty nanolipogels or nanolipogels co-encapsulatedwith SB505124 (SB) or IL-2. No hepatoxicity was observed, as measured byserum levels of alkaline phosphatase and alanine aminotransferase.Normal physiological reference ranges given by the IDEXX VetTest® systemfor mouse alkaline phosphatase and alanine aminotransferase were 62-209IU/L and 28-132 IU/L, respectively. Furthermore, no renal toxicity wasobserved, as blood urea nitrogen levels were within the normal mousereference range of 18-29 mg/dL. A complete blood count was alsoperformed to identify any hematological toxicity. Leukocyte counts,platelet counts, and hemoglobin content were all within normalphysiological ranges for mouse (leukocytes: 1.8-10.7×10³ cells/uL;platelets: 592-2971×10³ cells/uL; hemoglobin: 11.0-15.1 g/dL).Hematoxylin and eosin staining of lungs demonstrated no obviouspulmonary toxicity. Bronchiolar and alveolar structures appeared normal,and no disruption to epithelial layers or inflammatory infiltrates wereobserved in lung sections.

The in vitro results demonstrate that nanolipogels have similarnegligible toxicities to liposomes.

Healthy C57/B16 mice were administered a single intravenous dose ofnanoparticle combination therapy or controls and acute toxicologymeasured 7 days later. No significant toxicities were observed in serummeasurements of alkaline phosphatase or serum alanine aminotransferase.Normal physiological ranges for mouse alkaline phosphatase areapproximately 62-209 IU/L and for alanine aminotransferase approximately28-132 IU/L. No renal toxicity was observed, as blood urea nitrogenlevels were within the normal mouse reference range of 18-29 mg/dL.Complete blood counts demonstrated that normal physiological rangesleukocyte counts, platelet counts, and hemoglobin content. Lung toxicitywas evaluated by histology to determine presence of acute inflammation.Hematoxylin and eosin staining of lungs demonstrated no obviouspulmonary toxicity or inflammatory infiltrates; bronchiolar and alveolarstructures appeared normal with no disruption to epithelial layers.

Example 6: Drug Delivery to Tumors and Antitumor Activity—SubcutaneousTumors

The effects of IL-2 and TGF-β antagonist monotherapies to enhance theantitumor responses against B16 melanomas were determined.

Materials and Methods

A paired t test (two-tailed) was used to analyze differences in tumorareas and masses. OriginPro version 8.1 (Microcal) and Prism version5.01 (GraphPad Software, Inc.) were used for the analyses. Survivalstudies were analyzed using the Kaplan-Meier and Wilcoxon-Gehan testswith Origin.

In Vivo Subcutaneous Tumor Studies.

B16-F10 cells (ATCC) were cultured in DMEM (Gibco) and suspended at2×10⁶ cells/mL in 1×PBS (kept on ice) directly prior to injection. Forsubcutaneous tumor studies, female 6-8 week-old B6 albino mice weresedated with AErrane (isofluorane; Baxter) and the right hind flank wasshaved prior to a subcutaneous injection of 50 μL of the cellularsuspension. Tumors were monitored and treatment began when the averagetumor area reached approximately 5.5 mm² (8-10 days after B16 injection;mice were rearranged to normalize tumor sizes across groups). Mice weresedated with isofluorane for intratumoral nanolipogel administration.Each dose consisted of 5 mg nanolipogels.

Observers were blinded for tumor area and survival studies. Mice wereeuthanized with carbon dioxide when any one tumor dimension was greaterthan 15 mm, when exhibiting any sign of sickness, or at one weekpost-treatment for FACS analyses studies. The in vivo delivery studyused 5 mg injections/mouse of f-CD-solubilized rhodamine (Sigma)-loadednanolipogels, prepared as described above for nLG-SB. Five mice pergroup were euthanized at different timepoints and tumors were extractedand weighed.

Rhodamine was extracted by homogenizing the tumors in 500 μL deionizedwater (DI). Two freeze/thaw cycles at −80° C./room temperature were usedto ensure cells were completely lysed then homogenates were thawed toroom temperature and 40% (v/v) dimethylsulfoxide and 1% (v/v) TWEEN® 80were added to dissolve particles. After vortexing, the homogenates werefrozen at −80° C. for 24 hours, thawed at room temperature and vortexedfor 10 minutes, then cellular debris was pelleted by 30 minutescentrifugation at 13000 rpm. The supernatant was removed andfluorescence was measured with excitation 540 nm/emission 625 nm.

In Vivo Nanolipogel

As encapsulation in nanolipogels decreased clearance of free drug andimproved biodistribution, localized therapy of subcutaneous tumors wasevaluated first to assess therapeutic efficacy. Weekly intratumoraladministration of soluble SB alone failed to delay tumor growth (FIG.4A), consistent with previous results using LY364947 in preclinicalprostate and gastric animal cancer models. A similar null effect wasobserved when both soluble SB and IL-2 were co-administered in weeklydoses (FIG. 4A). The nanolipogel encapsulated SB administeredindividually (nLG-SB) significantly delayed tumor growth (FIG. 4A),resulting in mice with smaller tumors after one week of therapy (FIG.4B). Although nanolipogel encapsulated IL-2 administered individually(nLG-IL-2) did not significantly delay tumor growth (FIG. 4A), the tumormasses at one week were significantly smaller than tumors obtained frommice in the control group (FIG. 4B).

These results are in accord with prior studies demonstrating theefficacy of sustained release of either IL-2 or small moleculesinhibiting TGF-β signaling over pulses of these agents for enhancementof antitumor responses. When comparing all treatment groups, the moststriking and significant reduction in both tumor growth rate and tumormass after one week of therapy was observed in the mice receivingsimultaneous sustained delivery of SB and IL-2 (FIGS. 4A and 4B).

Results

Tumor size in treated and untreated mice correlated with their survival(FIG. 4A; 4C). Administration of soluble SB or SB in combination withIL-2 did not improve survival over untreated controls, while nanolipogelformulations of IL-2 or SB alone modestly increased average survivaltimes (FIG. 4C). In contrast, the nanolipogel-delivered combinationimmunotherapy dramatically increased survival (FIG. 4C). As observedwith the tumor kinetics data, administration of particles releasing eachagent slightly improved survival; however, mice receiving combinationtherapy via particles releasing both agents demonstrated markedlysmaller tumors and longer survival compared to the other treatmentgroups. Of the animals receiving nLG-SB+IL-2, 100% survived through thestudy endpoint at 35 days after initial tumor implantation. Completetumor regression and survival was observed in a cohort (40%) of thegroup through 60 days. (FIG. 4C)

Drug delivery to tumors following localized peritumoral injection ofrhodamine-loaded nLG (nLG-rhod) was evaluated by comparative measurementof rhodamine concentrations in tumors versus peritumoral tissues. Thepharmacokinetic profile suggested sustained delivery of drug from thelocalized depot of nLG: at 24 hours after peritumoral administration,only 3±1% of the initial dose had penetrated into the tumor mass and36±17% of the initial dose remained in the surrounding tissues. Over thecourse of 7 days, the cumulative rhodamine concentration in the tumorincreased to 25±0.5% of initial dose, while total rhodamineconcentration in the peritumoral tissue decreased to 4±2% of initialdose.

Example 7: In Vivo Nanolipogel Biodistribution, Safety, and MetastaticLung Tumor Studies

A significant unmet need is improved biodistribution of short-livedcytokines and hydrophobic drugs in the treatment of distant metastatictumors. Genetically modified mice containing T-cells resistant to TGF-βsignaling abrogated the development of metastatic B16 melanoma depositsin the lungs, providing additional motivation for assessing TGF-βblockade with and without IL-2 therapy in this tumor model. The effectof systemic nanolipogel therapy against highly aggressive B16 lungmetastases was tested in this model. It has previously been shown thatintravenous injection of $16 cells results in rapid metastatic tumorgrowth in the lungs of B6 mice.

Materials and Methods

Metastatic B16 melanomas were established in female 6-8 week old B6 miceby intravenous (tail vein) administration of 50 μL of B16 cellularsuspension as described by Gorelik, et al. Nat Med 7, 1118-1122 (2001).Treatment was initiated 7 days later with each dose consisting of 5 mgnanolipogels administered intravenously via tail vein injection. Micewere euthanized when exhibiting external tumor growths, paralysis orweakness, significant weight loss, or at 14 days after the firsttreatment for FACS analyses studies. After sacrifice, the chest cavitywas exposed and the lungs perfused by making a small incision in theright atrium and injecting 10 mL of cold PBS into the right ventricle.Lung metastates were counted by blinded observers who recorded darkcircular masses ranging from 0.1-3 mm as unique tumors.

Biodistribution studies of nanolipogels were conducted in healthy andtumor-bearing mice after injection (local or systemic) of 5 mg off-CD-solubilized rhodamine-loaded nanolipogels (with or withoutfluorescein-labeling of the lipid carrier). Rhodamine was extracted fromhomogenized tissues and quantified as described above. Acute toxicologywas assessed in healthy C57/B16 mice seven days after an intravenousdose of buffer control, empty nanolipogels, or SB and IL-2-loadednanolipogels. Hepatoxicity and renal toxicity were assessed viameasurement of serum alkaline phosphatase, alanine aminotransferase andblood urea nitrogen levels. A complete blood count was performed toidentify any hematological toxicity. Finally, lung toxicity wasevaluated by histology to determine if systemically administerednanolipogels induced any acute inflammation.

Resected tumor tissue was fixed in 10% formalin for 24 hours and thenembedded in paraffin. Tissue blocks were sectioned into 5 um slices andmounted on glass slides followed by hematoxylin and leukocyte staininganti-LCA (CD45)-peroxidase conjugate (1 ug/ml) (Life technologies).

An Olympus BX61WI fluorescence microscope in combination with a 20×,0.95NA Olympus objective and LaVision Biotec two-photon microscopysystem was used for imaging tumor vasculature and nanolipogelaccumulation in tumors. Briefly, incisions were made to expose skinflaps surrounding subcutaneous tumors on anesthetized C57BL/6 mice.Intravital image acquisition was started 5 minutes after intravenousadministration of nanolipogels. An auto-tunable Titanium-Sapphiretwo-photon laser (Chameleon Vision II, Coherent) pumped by a Verdi lasersource was used for the excitation light source. Emitted light wascollected with non-descanned detectors outfitted with the followingbandpass filters: 435/90 nm, 525/50 and 615/100. The field of view foreach xy plane was either 400 μm×400 μm or 500 μm×500 μm, at a resolutionof 0.8 um per pixel. Stacks of between 26 and 101 optical sections with1 or 2 μm z spacing were acquired every 60 s over the course of one hourwith the laser set at a wavelength of either 850 nm or 940 nm. Volocity®software (Improvision) was used to create sequences of image stacks.

Results

Tumor infilitrating lymphocytes were isolated from B16 melanomas asdescribed by Petersen, et al. J Immunother 29, 241-249 (2006).Treatments were given by tail vein injection and were initiated one weekafter cell injection to assess efficacy against growing tumors. As wasthe case in mice bearing subcutaneous tumors, maximum survival benefitwas observed in the group receiving the nanolipogel-encapsulatedcombination therapy. Mantel-Cox analysis demonstrated a statisticallysignificant (p<0.01) increase in survival over animals receiving salinealone, i.e., no treatment (Figure Sa). Half of the animals receivingnLG-SB+IL-2 survived through the study endpoint at 45 days. (FIG. 5a )

To examine the effect of treatment on tumor burden, animals weresacrificed two weeks after initial treatment and whole lung samplesinspected visually for melanoma deposits. Administration of soluble SBwith or without IL-2 co-therapy failed to reduce the number of lungtumors at three weeks (FIG. 5b ). Maximum tumor burden reduction wasobserved in animals receiving the nanolipogel-delivered combinationtherapy (FIG. 5b ).

Comparative biodistribution was repeated in mice bearing B16 metastaticlung tumors. Dual-labeled nanolipogels formulated by incorporatingfluorescein-labeled PEG-phosphoethanolamine into the lipid membrane ofrhodamine-loaded nanolipogels were used to assess trafficking of theparticles versus trafficking of the payload. Fluorescein-labeled PEG didnot interfere with detection or release of rhodamine. Lung tumor-bearingmice received a single IV (tail vein) dose of dual-labeled nanolipogel.B16 metastases were often visible as approximately 1 mm irregularnodules under bright field observation while the fluorescein-labeledlipid of the delivery vehicle and rhodamine were detected underfluorescent filters up to 24 hours post administration. Fluorescentdetection of lipid was significantly diminished by 4 days afteradministration.

To test for accumulation of nanolipogel and drug in distant tumorsbeyond lung tissue, biodistribution experiments were repeated in micebearing distant subcutaneous tumors. Following intravenous injection,lipid and rhodamine concentrations were quantified in subcutaneoustumors and in homogenized tissues at various time points afteradministration. Peak tumoral concentrations of lipid and rhodamine,8.8±4.0% and 2.5±0.8% per gram of tumor, respectively, were observed oneday after administration. An analysis of all tissues confirmedbiodistribution patterns that were similar for both the lipid andrhodamine components of the nanolipogel system indicating that the drugpayload was associated with the particles during biodistribution.Trafficking of nanolipogels and payload within the vasculature of tumorswas validated by imaging subcutaneous tumors with time-resolvedtwo-photon laser scanning intravital microscopy after intravenousinjection. Accumulation of fluorescein-labeled nanolipogels along thevasculature was detected both in the areas surrounding tumors as well aswithin the tumor itself within 30 minutes post-intravenous injection.Particle trafficking in the tumor vasculature was accompanied by anincrease in the encapsulant fluorescence in the tumor microenvironment.Extravasation of rhodamine was evident in peritumoral tissue as well asthe interstitial spaces between tumor cells.

Fluorescein-labeled PEG did not interfere with detection or release ofrhodamine. An analysis of all tissues confirmed biodistribution patternsthat were similar for both the lipid and rhodamine components of thenanolipogel system as well as within the tumor itself within 30 minutespost-intravenous injection.

Example 8: Immunological Mechanisms of Antitumor Effects of Nanolipogels

Materials and Methods

In order to elucidate the immunologic mechanisms behind the therapeuticeffects of the sustained release combination therapy and the relativecontribution of each agent delivered as monotherapies, tumorinfiltrating lymphocytes (TILs) were harvested and evaluated in miceeuthanized one to two weeks after the initial therapeutic dose. Thistime point was chosen based on when mice in all groups had developedeither subcutaneous tumors of sufficient size (up to 10 mm in greatestdimension), or sufficient numbers of lung tumors (more than five), toisolate adequate number of TILs for analysis.

Tumor infiltrating lymphocytes were isolated from B16 melanomas. Inbrief, subcutaneous tumors or lung tumors were resected from mice,weighed, minced into sections approximately 3 mm in greatest dimension,then placed in 8 mL of serum-free RPMI media (Irving Scientific SantaAnna, Calif.) containing 175 U/mL of Collagenase IA (Sigma, no. C9891)for subcutaneous tumors or 100 U/mL of Collagenase IV (MP Biomedical,no. 195110) for lung tumors. The resulting tissue suspension wasincubated at 37° C. for 1 hour, passed through a 70-pin tissue filterand the resulting cells were washed twice in serum free RPMI media. Thepellet was resuspended in 0.5 mL of RPMI media then overlaid over mouselympholyte-M media (Accurate Chemical, Westbury, N.Y.) for lymphocyteisolation, followed by centrifugation at 1500×g per the manufacturer'sinstructions (Accurate Chemical). The resulting buffy coat layer wasremoved and washed in RPMI media as described above and subsequentlyresuspended in 1 mL of 1×PBS containing 0.5% bovine serum albumin(Sigma). All cell suspensions were counted to determine absolute numbersof isolated TILs and subsequently distributed to 96 well-plates for FACSstaining and analysis.

A Becton Dickenson LSRII flow cytometer was used to quantify thepercentage of immune effector cells (CD4⁺, CD8⁺ T-cells and NK cells) aswell as Tregs in the tumor-bearing mice by evaluating the cell surfaceexpression of CD4, CD8, NK1.1, TCR-beta as well as intracellular FoxP3as per manufacturer's instructions (eBioscience, San Diego, Calif.).Absolute cell numbers were assessed by direct counting on a Coulter cellcounter. Anti-CD4 (Pacific Blue-conjugated, no 558107) anti-CD8-a(Peridinin-chlorophyll-protein (PercP) conjugated, no. 553036),anti-NK1.1 (Fluoroscein isothyocyanate (FITC) conjugated, no. 553164),anti-TCR-beta (allophyocyanin (APC)-conjugated, no. 553174), anti-CD44(FITC-conjugated, no. 553133) and anti-CD62L (APC-conjugated, no.553152) were purchased from Becton Dickenson Pharmingen. The anti-FoxP3kit (containing phycoerytherin-conjugated FoxP3 antibody, no.72-5775-40) was purchased from eBioscience.

Cells were incubated with 40 μL antibody cocktails diluted appropriately(per vendor instructions) in IX PBS containing 0.5% BSA and a 1:200dilution of Fe blocking antibody 2.4G2 (anti-CD16/32) to preventnonspecific binding. Cells were incubated with the antibody cocktailsfor 30 minutes at 4° C. then washed once prior to analysis on LSRIIGreen flow cytometer. For intracellular staining of FoxP3, cells werefixed, permeabilized and stained using a FoxP3 staining kit purchased(eBiosciences).

All FACS data were analyzed using FloJo software (Tree Star Inc.,Ashland, Oreg.). Absolute CD4 Tregs, CD8 T-cells and NK cells weredetermined by multiplying the absolute numbers of TILs normalized pergram of tumor by the percentages (gated first on live, TCR-β+/−) ofCD4⁺/FoxP3⁺, CD8⁺ or NK1.1⁺/TCR-β cells measured by the LSRII Green flowcytometer. CD8/Treg ratios were obtained by dividing the absolute numberof CD8 T-cells by the absolute number of regulatory T-cells.

NK1.1 antibodies were isolated from HB191 hybridoma as described byYokoyama, W. M. Monoclonal antibody supernatant and ascites fluidproduction, in Curr Prot Immunol (ed. Margulies, D. H.) 2.6.1-2.6.9(John Wiley & Sons, New York, 2000). These antibodies were injectedintraperitoneally one day prior to B16 injection and every seven daysthereafter. Each injection was 250 μL of a 1 mg/ml solution.

To confirm the NK depletion occurred successfully in mice treated withthe NK1.1 antibody, approximately 300 microliters of blood and thespleens of NK depleted and NK proficient mice were obtained. Peripheralblood and splenocytes which were obtained using mechanical dissociationwere treated with ACK lysis buffer (Lonza, Walkersville, Md.) as permanufacturers protocol and subsequently stained for NK1.1 or TCR-β usingthe antibodies described in the text. Percentages of NK1.1/TCR-betanegative cells indicated in right lower quadrants in Supplementary FIG.8, which show successful NK depletion with anti-NK1.1 antibodies.

Results

The effects of IL-2 and TGF-β antagonist monotherapies to enhance theantitumor responses against B16 melanomas were evaluated. Asencapsulation in nanolipogels decreased, clearance of free drug andimproved biodistribution, resulting in mice with smaller tumors afterone week of therapy, the tumor masses at one week were significantlysmaller than tumors obtained from mice in the control group.

Administered in nanolipogels alone or in combination with SB, IL-2increased both the percentage and absolute numbers of activated CD8⁺T-cells in tumors (FIG. 6a ; FIG. 6b ) with minimal impact on overallCD4/CD8 ratios and T_(regs), results that were consistent with reportedclinical outcomes. Representative histological images of tumors showedthat IL-2 significantly increased lymphocyte infiltration into tumors.Sustained administration of this cytokine also increased activatedCD8⁺:T_(reg) ratios in TIL populations. (FIG. 6c )

Treatment with nLG-SB significantly increased activated CD8⁺ populations(P<0.05), as did treatment with nLG-IL-2 or nLG-SB+IL-2 (P<0.001), overunloaded particles (nLG-Empty). All groups have significantly greaterratios (P<0.05) compared with empty nLGs.

These data, however, did not fully explain the observed results in micereceiving particles releasing both IL-2 and SB, suggesting anothermechanism may be involved in the enhanced antitumor effects observed inthe treated mice. Since TGF-β can also regulate NK cell number andfunction, it was assessed whether this cell type was involved in theinnate arm of the immune system present in TILs. In stark contrast tothe relative number of TIL T_(regs) observed in all tumor bearing mice,sustained administration of SB in combination with IL-2 resulted insubstantially increased percentage (FIG. 6a ) and absolute numbers (FIG.6c ) of NK cells present in tumor beds compared to groups receivingeither “empty” particles or particles releasing either IL-2 or SB alone.

To validate that the therapeutic benefit observed in mice treated withparticles releasing both agents was NK-dependent, studies were performedin NK-depleted mice. NK1.1 antibodies were used to deplete mice of NKcells and tumor cells were injected in NK-depleted mice and miceretaining NK cells. Mice were again euthanized one week after theinitial treatment and tumor masses were measured.

Compared to the empty particle group, significantly more NKs werepresent in the lungs following treatment by nLG-SB+IL-2 (P<0.05), nLG-SB(P<0.05), and nLG-IL-2 (P<0.01). The nLG-SB+IL-2-treated group hassignificantly more NKs than the control group (P<0.01), the SB-treatedgroup (P<0.05), and the IL-2-treated group (P<0.01). ThenLG-SB+IL-2-treated WT group has significantly smaller tumors than allother treatment groups (P<0.001). The NKD nLG-SB and nLG-SB+IL-2 groupshave significantly larger tumors than their WT counterparts (bothP<0.001). Studies were repeated 2-3 times with similar results.

NK depletion did not affect the sizes of tumors in mice receivingparticles releasing IL-2 alone (FIG. 6b ). In contrast, absence of thesecells abrogated the delay in tumor growth in animals receiving particlesreleasing SB and IL-2 (FIG. 6b ). There was a modest therapeutic benefitin mice receiving particles releasing drug alone that was abrogated byNK depletion (FIG. 6b ). There was a modest, statistically significantincrease in NK cells in the TILs of mice treated with particlesreleasing SB alone (FIG. 6c ).

Thus, the maximum therapeutic benefit observed in mice treated withparticles simultaneously delivering SB and IL-2 therapies was likelyrelated to enhanced numbers of NK cells at the tumor site, resulting inincreased effector cell populations in the tumor.

Importantly, the clinical effects following systemic therapy wereconsistent with the results of localized therapy and drugbiodistribution. Encapsulation in nanolipogel increased both the initialdose to the lungs as well as dose persistence over a three day period;on the third day after administration, 9.0±0.8% (mean±s.d.) of theinitial dose of nanolipogels was measured in the lungs compared to1.5±0.7% of soluble drug. This pharmacokinetic effect correlates withincreased survival and a significant decrease in the number of tumors.Analysis of lung-infiltrating lymphocytes demonstrated that, as observedin subcutaneous tumors receiving intratumoral nLG-SB+IL-2 treatments,enhanced numbers of activated CD8⁺ (FIG. 5a ) and NK (FIG. 6a ) effectorcells mediated tumor abrogation and increased survival. These dataindicate that significant antitumor responses against metastaticmelanoma can indeed be achieved by the sustained, combined delivery of aTGF-β inhibitor drug and IL-2 in a clinically-relevant mode ofadministration.

Example 9: Comparative Distribution of Nanolipogel Carrier andEncapsulant

Materials and Methods

The distribution of both nanolipogel carrier and encapsulated drugpayload was investigated using dual-labeled nanolipogels.Fluorescein-labeled phosphoethanolamine was incorporated into the lipidcomponent of rhodamine-loaded nanolipogels. Spectrofluorimetric analysisat 540/625 nm and 490/517 nm demonstrated a dose-dependent fluorescencewith no spectral overlap. There was controlled release of rhodamine, butnot lipid.

Mice bearing subcutaneous B16 tumors received a single IV (tail vein)injection of dual-labeled NLG. Animals were sacrificed at 1, 2, 3, and 7days post injection and tissues collected for homogenization,extraction, and quantification of rhodamine and fluorescein-PE.

Results

Analysis of serum showed prolonged circulation of both encapsulant anddelivery vehicle. Similar patterns of biodistribution were observedbetween lipid and drug payload, with highest accumulations of drugoccurring in the lungs and liver.

Example 10: Lipid Encapsulated Dendrimers for Combined Delivery ofNucleic Acids, Proteins, and Drugs

The nanolipogel encapsulating dendrimers includes a main shellconsisting of a liposome encapsulating a drug and siRNA/dendrimercomplex which inserts in cells. Dendritic polymers (dendrimers) are aclass of monodisperse polymers distinguished by their repeated branchingstructure emanating from a central core. This branching, which isinherent in the divergent synthesis of dendrimers, leads to a geometricgrowth of the polymer that can nearly approximate a sphere withincreased branchings or higher generations (generation 6 or above). Thisbranching creates a core ideally suited for entrapment of a variety ofsmall hydrophobic molecules such as drugs as well as complexation ofnucleic acids. For example, SUPERFECT® is a commercially availableactivated dendrimer transfection agent. Combined with their narrowmolecular weight distribution and small size (less than 10 nm),dendrimers have been utilized for a large number of applicationsincluding drug and gene delivery. Dendrimers complexed with nucleicacids can be cleared rapidly upon in vivo administration and henceprotective targeting of this complex would be a more attractive modalityfor site-specific delivery. The liposomal formulation serves twofunctions: 1) Protective encapsulation of siRNA complexed liposomes and2) facilitating delivery of small molecule hydrophilic drugs such asrivoavrin or proteins such as IFNα. Complexation of the inner dendrimercore with siRNA: Nucleic acids are generally stabilized by cationicpolymers in a polyplex formation akin to the physiological packaging ofnucleic acids around histones. Cationic polyamidoamine (PAMAM)dendrimers, generation 5 (G5), diameter 5.4 nm, serves this purpose.

Materials and Methods

siRNA/Dendrimer polyplexes are formed by combining G5 PAMAM and siRNA atan amine to phosphate (N/P) ratio of 1:1 to 10:1. The precise ratiowhich will yield optimal silencing can be determined by mixing stocksiRNA and PAMAM at different molar ratios for 30 min at room temperaturein sterile 10 mM HEPES buffer, pH 7.2 with light vortexing. Thisprocedure yields a siRNA-dendrimer polyplex with a charge (zetapotential) of +20 or above and effective diameter of 10 nm, which issuitable for encapsulation in liposomes. Next, the polyplex isco-encapsulated with the drug (IFNα and/or Ribavirin) in the liposomalparticle. A dehydrated lipid film comprised ofdistearoyl-glycero-phosphocholine (DSPC), cholesterol, anddistearoylglycero-phosphoethanolamine (DSPE) with an amine terminatedpolyethylene glycol (PEG2000) spacer (DSPE-PEG2000-NH2) is first mixedin the molar ratio of 65:30:5, then rehydrated under sonication with a10 mg/ml solution of siRNA/Dendrimer polyplex and drug. The ratio ofdrug to siRNA/dendrimer in solution can be tuned during formulation.Optimal ratio is dictated by in vitro and in vivo efficiacy studies. Theintrinsic “built-in” lipid PEGylation facilitates a longer circulationtime compared to particles without PEG. PEG incorporation yields asteric hydration barrier shield which facilitates long-lived in vivocirculation. (i.e avoidance of the reticuloendothelial system andnon-specific uptake by macrophages).

Following the mixing of drug and siRNA/Dendrimer polyplex in presence oflipids, the solution is extruded through a series of filters. Firstthree times through a 5 μm filter, three times through 1 μm filter, andfive times through a 200 nm filter collecting extrudate in a steriletube. Excess siRNA complex, drug and lipids are removed by spinning for45 minutes at 24000 rpm at 4 C (3×) in an ultracentrifuge.

FIG. 7A is a schematic of LED preparation encapsulating siRNA/dendrimerpolyplex and drug combinations, with covalent modification of the outershell with targeting antibodies or single chain variable fragments(scFv). Attachment of antibodies or scFv to the amine terminatedliposome is achieved by activating the protein in 0.1 MES buffer (pH5.5) in the presence of ethyldicarbodiimide and N-hydroxysuccinimide for10 min followed by addition to particles in buffered saline (pH 7.4).This reaction activates carboxylate groups on the protein for covalentlinkage to exposed amine groups on the particles (ref). Initially, thereaction stoichemetery is adjusted to yield an approximate density of1-10 scFv molecules per particle, however, this density can be easilyincreased by varying the stoichiometry of the reaction to facilitatemaximal internalization. Total time for the reaction is 30 min at roomtemperature. These reaction conditions have no effect on the integrityor function of encapsulated agents.

LEDs were tested for the ability to deliver a functional expressionvector (pGFP) into BMDC, HeLa, 293T cells using differentmethacrylate-conjugated β-cyclodextrins (CDs) and Nitrogen/Phosphorus(NIP) ratio, and compared to vector delivery using LIPOFECTAMINE® 2000and liposomes.

LEDs were also tested for the ability to deliver functional siRNA.Jurkat (human T cell line) were incubated with LEDs encapsulating siRNAagainst CD4 or Luciferase (Luc) and surface functionalized with anti-CD7to mediate internalization.

Results

LEDs can facilitate drug internalization as depicted in FIG. 7A withmacrophages in culture. The drug Methotrexate (MTX) was used as a modeldrug. FIG. 7B shows the cytotoxicity of LED and LED encapsulating themodel drug methotrexate (MTX). Bars indicate successive dilutions of LEDor drug or combinations starting from (1 mg/ml left to right to 10ug/ml). Azide is used as a positive control for cell killing. Startingat 10%, left to right, and increasing to 1%, the graph shows that,compared to free drug (MTX), LED containing MTX were slightly lesstoxic, presumably because of drug sequestration. LEDs alone showed nocytotoxicity.

LEDs encapsulating the dye rhodamine facilitate internalization andcytoplasmic localization of the dye and LEDs containing the pGFP plasmidshowed enhanced efficiency in transfection of macrophages compared to astandard transfection agent such as LIPOFECTAMINE®.

To determine if terminal amine groups on PAMAM dendrimers provideendosomal buffering and disrupt endosomes by the proton sponge effect,an Acridine Orange (a dye whose spectral properties change depending onits location in endosomes or cytosol) assay was used with BMDCs, whichwere treated with unmodified generation 4 PAMAM dendrimers (G4), ordendrimers conjugated to cyclodextrin molecules (CD) that substitutedand shielded primary amines with or without ionophorecarbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The resultsindicate that of the tested combinations, unmodified G4 dendrimer wasbest at endosomal disruption followed by G4-3CD (FIG. 7C). G4-6CD wasthe least effective at endosomal disruption of the combinations tested,supporting the idea that proton sponge effect is mediated by primaryamines and substituting amines with CD decreases buffering capabilities.

LEDs were also tested using different dendrimer (G)-cyclodextrinconjugates (CDs) and Nitrogen/Phosphorus (N/P) ratio, and compared tovector delivery (pGFP) using LIPOFECTAMINE® 2000 and liposomes in avariety of cells types. CD significantly affected dendriplex (FIG. 7D).Dendriplexes (from modified dendrimers) transfect better thanLipofectamine 2000 in BMDCs. LEDs also transfected better than liposomesencapsulating vector in BMDCs.

LEDs encapsulating siRNA against CD4 or Luciferase (Luc) and surfacefunctionalized with anti-CD7 were test for the ability to mediateinternalization target mRNA knockdown in Jurkat (human T cell line)cells. Results indicated that LED delivered siRNA reduced surfaceexpression of CD4, or Luc relative to controls (FIG. 7E).

In a second experiment, LEDs including 200 ug of dendrimer and 400 pmolsiGFP (Ctl=LFA:siGFP) were utilized to knockdown GFP expression is astably transfected cell line. Stable 293T-eGFP cells were treated withdendrimers for 4 h in SFDMEM followed by examination of GFP expression.Cells treated with most dendrimer (G)-cyclodextrin conjugates (CDs)combinations exhibited greater reduction in GFP expression compared tomock and LIPOFECTAMINE®:siGFP controls (FIG. 7F).

Example 11: Antigen Cross Presentation with Lipid EncapsulatedDendrimers

Materials and Methods

Mouse Bone Marrow-Derived Dendritic Cells (BMDCs) were incubated withliposomes encapsulating ovalbumin (OVA) alone, dendrimer alone, or bothOVA and dendrimer (LED).

Results

Controls of cells and empty liposomes showed undetectable levels of25.D16 antibody staining, which binds MHC Class I-SIINFEKL complexes.Cells receiving LEDs showed the highest level of antigencross-presentation. * p<0.05 by one-way ANOVA Bonferroni post-test. (SeeFIG. 8A).

Example 12: Vaccine Delivery with Lipid Encapsulated Dendrimers

Materials and Methods

Antigen Presentation

1×10⁵ BMDC/well (96 well plate)+25 uL liposomal particles. Particlegroups:

-   -   a. −/− (nothing outside nothing inside particles)    -   b. −/OVA (nothing outside, OVA encapsulated)    -   c. −/G5+OVA (nothing outside, OVA and G5 dendrimer inside)    -   d. −/G5+OVA+CpG    -   e. MPLA/− (MPLA outside, nothing inside)    -   f. MPLA/OVA    -   g. MPLA/OVA+G5    -   h. MPLA/OVA+G5+CpG (MPLA outside; OVA, G5 dendrimer, CpG        encapsulated)

Where OVA=ovalbumin, MPLA=monophosphoryl lipid A, G5=generation 5dendrimer, CpG=CpG oligonucleotide (TLR9 ligand).

Treatment was incubated with BMDC for 24 hours followed by 4 dayco-incubation with WT splenocyte.

Analysis of Pro-Inflammatory Cytokine Production

BMDCs were incubated with liposomal nanoparticles encapsulating antigenand surface-functionalized with increasing amounts of TLR ligand CpG for24 hours before supernatant analysis by ELISA.

Results

Cells were stained with 25.D16-PE, an antibody that is specific formouse MHC Class I-SIINFEKL complexes, as assessed for antigencross-presentation by flow cytometery. The results indicated that −/OVAparticles induce some cross-presentation, which was increased by OVAparticles containing dendrimer. Particles combining MPLA, CpG, anddendrimer induce the highest amount of cross-presentation. (See FIG.8B). Liposomal nanoparticles surface functionalized with CpG alsoinduced a dose-dependent increase the production of pro-inflammatorycytokine IL-6. (See FIG. 8C, comparing blank particles, 0.1 μg CpG/mgparticles, 0.25 μg CpG/mg particles, and 0.5 μg CpG/mg particles.).

Modifications and variations of the compositions and methods ofmanufacture and use thereof will be obvious to those skilled in the artfrom the foregoing detailed description and are intended to come withinthe scope of the appended claims. All references are specificallyincorporated.

We claim:
 1. A nanolipogel comprising a polymeric matrix core comprisinga host molecule that can bind a therapeutic, diagnostic or prophylacticagent to be delivered, releasing the agent under in vivo conditions, anda lipid shell.
 2. The nanolipogel of claim 1 wherein the polymericmatrix, the lipid shell, or both are crosslinked.
 3. The nanolipogel ofclaim 1 comprising an agent complexed to the host molecules, dispersedwithin the polymeric matrix, dispersed in or bound to the lipid shell,or combinations thereof.
 4. The nanolipogel of claim 1 wherein thepolymeric matrix comprises polymer selected from the group consisting ofpolylactic acid), poly(glycolic acid), poly(lactic acid-co-glycolicacids), polyhydroxyalkanoates; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);poly(glycolide-co-caprolactones); polycarbonates; polyamides,polypeptides, and poly(amino acids); polyesteramides; otherbiocompatible polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophilic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polysiloxanes; poly(oxyethylene)poly(oxypropylene)copolymers; polyketals; polyphosphates; polyhydroxyvalerates;polyalkylene oxalates; polyalkylene succinates; poly(maleic acids),polyvinyl alcohols, polyvinylpyrrolidone; poly(alkylene oxides);celluloses, polyacrylic acids, albumin, collagen, gelatin, prolamines,polysaccharides, derivatives, copolymers, and blends thereof.
 5. Thenanolipogel of claim 3 wherein the agent is selected from the group oftherapeutic, prophylactic, diagnostic, and nutraceutical agentsconsisting of small molecule active agents, proteins, polypeptides,polysaccharide, and nucleic acids.
 6. The nanolipogel of claim 5 whereinthe agent is selected from the group consisting of antibiotics,antivirals, anti-parasitics, cytokines, growth factors, growthinhibitors, hormones, hormone antagonists, antibodies and bioactivefragments thereof, antigen and vaccine formulations,anti-inflammatories, immunomodulators, and oligonucleotide drugs,paramagnetic molecules, fluorescent compounds, magnetic molecules, andradionuclides, x-ray imaging agents, and contrast agents.
 7. Thenanolipogel of claim 6 wherein the agent is selected from the groupconsisting of alkylating agents, antimetabolite, antimitotics,anthracyclines, cytotoxic antibiotics, topoisomerase inhibitors,antibodies to vascular endothelial growth factor; thalidomide;endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors);tyrosine kinase inhibitors; transforming growth factor-α or transforminggrowth factor-β inhibitors, and antibodies to the epidermal growthfactor receptor.
 8. The nanolipogel of claim 1 comprising a liposomalshell composed of one or more concentric lipid layers, optionallycrosslinked, wherein the lipids can be neutral, anionic or cationiclipids at physiologic pH.
 9. The nanolipogel of claim 8 wherein thelipid is selected from the group consisting of cholesterol,phospholipids, lysolipids, lysophospholipids, and sphingolipids, andderivatives thereof.
 10. The nanolipogel of claim 8 comprising lipidselected from the group consisting of phosphatidylcholine;phosphatidylserine, phosphatidylglycerol, phosphatidylinositol;glycolipids; sphingomyelin, ceramide galactopyranoside, gangliosides,cerebrosides; fatty acids, sterols;1,2-diacyl-sn-glycero-3-phosphoethanolamines,1,2-dihexadecylphosphoethanolamine, 1,2-distearoylphosphatidylcholine,1,2-dipalmitoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine,N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium salts,dimethyldioctadecyl ammonium bromide, 1,2-diacyloxy-3-trimethylammoniumpropanes, N[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine,1,2-diacyloxy-3-dimethylammonium propanes,N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride,1,2-dialkyloxy-3-dimethylammonium propanes,dioctadecylamidoglycylspermine,3-[N-(N′,N-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol);2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminiumtrifluoro-acetate (DOSPA), β-alanyl cholesterol, cetyltrimethylammoniumbromide (CTAB), diC₁₄-amidine,N-tert-butyl-N′-tetradecyl-3-tetradecylamino-propionamidine,N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG),ditetradecanoyl-N-(trimethylammonio-acetyl) diethanolamine chloride,1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), andN,N,N′,N′-tetramethyl-, N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide,1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives, and 2,3-dialkyloxypropyl quaternary ammoniumderivatives containing a hydroxyalkyl moiety on the quaternary amine.11. The nanolipogel of claim 1 wherein the lipid is a PEGylatedderivative of a neutral, anionic, or cationic lipid.
 12. The nanolipogelof claim 10, wherein the1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives are selected from the group consisting of1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM) and1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM).
 13. The nanolipogel of claim 10, wherein the2,3-dialkyloxypropyl quaternary ammonium derivatives containing ahydroxyalkyl moiety on the quaternary amine are selected from1,2-dioleoyl-3-dimethyl[1]hydroxyethyl ammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),1,2-dioleyloxypropyl-3-dimetyl-hydroxypropyl ammonium bromide(DORIE-HP), 1,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammoniumbromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE[1]Hpe),1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).
 14. The nanolipogel of claim 4, wherein thepolyhydroxyalkanoates are selected from the group consisting ofpoly3-hydroxybutyrate and poly4-hydroxybutyrate.